Gene Expression in Recombinant Microorganisms (Biotechnology and Bioprocessing Series)

GENE EXPRESSION IN RECOMBINANT MICROORGANISMS

BIOPROCESS TECHNOIX)GY Series mitor

W. Courtney McGregor Xoma Corporation Berkeley, California

1. Membrane Separations in Biotechnology, edited by W. Courtney McGregor 2. Commercial Production of Monoclonal Antibodies: A Guide for ScaleUp, edited by Sally S. Seaver 3. Handbook on Anaerobic Fermentations, edited by Larry E. Erickson and Daniel Yee-Chak Fung 4. Fermentation Process Developmentof Industrial Organisms, edited by Justin 0. Neway 5. Yeast: Biotechnology and Biocatalysis, edited by Hubert Verachtert and Rend De Mot 6. Sensors in Bioprocess Control, edited by John V. Twork and Alexander M. Yacyn ych 7. Fundamentals of Protein Biotechnology, edited by Stanley Stein 0. Yeast Strain Selection, edited by Chandra J. Panchal 9. Separation Processes in Biotechnology, edited by Juan A. Asenjo 10. Large-scale Mammalian Cell Culture Technology, edited by Anthony S. Lubiniecki 11. Extractive Bioconversions, edited by Bo Mattiasson and Olle Holst 12. Purification and Analysis of Recombinant Proteins,edited by Ramnath Seetharam andSatish K. Sharma 13. Drug Biotechnology Regulation: Scientific Basis and Practices, edited by Yuan- yuan H. Chiuand John L. Gueriguian 14. Protein Immobilization: Fundamentals and Applications, edited by Richard F. Taylor 15. BiosensorPrinciples and Applications, edited by Loi'c J. Blum and Pierre R. Coulet 16. Industrial Application of Immobilized Biocatalysts, edited by Atsuo Tanaka, Tetsuya Tosa, and Takeshi Kobayashi 17. Insect Cell Culture Engineering, edited by Mattheus F. A. Goosen, Andrew J. Daugulis, and Peter Faulkner 18. Protein Purification Process Engineering, edited by Roger G. Harrison 19. Recombinant Microbes for Industrial and Agricultural Applications, edited by Yoshikatsu Murooka and Tada yuki lmanaka 20. Cell Adhesion: Fundamentals and Biotechnological Applications, edited by Martin A. Hjortso and Joseph W. Roos

edited by Juan A. Asenjo and Josd C. Merchuk 22. GeneExpressioninRecombinantMicroorganisms, edited by Alan Smith

21. Bioreactor System Design,

ADDITIONAL VOLUMES IN PREPARATION

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GENE EXPRESSION IN RECOMBINANT MICROORGANISMS editedby

Alan Smith

CIBA-GEIGY AG Basel, Switzerland

MarcelDekker, Inc.

NewYork.Basel

HongKong

Library of Congress Cataloging-in-Publication

Data

Gene expression in recombinant microorganisms le d i t e d by Alan Smith. p.cm. - (Bioprocesstechnology ; 22) Includes bibliographical references and index. ISBN 0-8247-9543-1 1. Microbialgenetics.2.Recombinantmicroorganisms.3.Gene expression.4.Geneticengineering-Methodology.I.Smith,Alan. II. Series. QH434.G44 1994 660’.65-d~20

94-35435

CP The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the address below. This book is printed on acid-free paper. Copyright 6 1995 by Marcel Dekker, Inc. All Rights Reserved. Neither this book nor anypart may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, orby any information storage and retrieval system, without permission in writing from the publisher. Marcel Dekker, Inc. 270 Madison Avenue, New York, NewYork10016 Current printing (last digit): l 0 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATES OF AMERICA

Series Introduction

Bioprocess technology encompasses all ofthe basic and applied sciences as well as the engineering requiredto fully exploit living systems and bring their productsto the marketplace. The technology that develops is eventually expressed in various methodologies and types of equipment and instruments builtup along a bioprocess stream. Typically in commercial production, the streambegins at the bioreactor, which can bea classical fermentor, acell culture perfusion system, or an enzyme bioreactor. Then comes separation of the product from the living systems and/or their components followed by an appropriate number of purification steps. The stream ends with bioproduct finishing, formulation, and packaging. A given bioprocess stream may have some tributaries or outlets and may be overlaid with a variety of monitoring devices and control systems. As with any stream, it will both shape and be shaped with time. Documenting the evolutionary shaping of bioprocess technology is the purpose of this series. Now that several products from recombinant DNA and cell fusion techniques are on the market, the new era of bioprocess technology is well established and validated. Books of this series represent developments ili

iv

Series Introduction

in various segments of bioprocessing that have paralleled progress in life the sciences. For obvious proprietary reasons, some developments in industry, although validated, maybe published only later,if at all. Therefore, our continuing series willfollow the growthof this fieldas it is available from both academia and industry. W. Courtney McGregor

Preface

The last decade has witnessed a remarkable growth inthe development of recombinant DNA technology. Applications include the use of genetically engineered microorganisms for the productionof biopharmaceuticals such as insulinand interferon; the early diagnosis of disease; gene therapy for inherited diseases; and the creation of transgenic animals for food production, for production of therapeutics, and for use in screening for new biologically active substances. Agricultural applications include the development of new plant strains, of plants resistant to pests and pesticides, and the deliberate release of engineered organisms for crop protection. The technology has also been exploitedto further our understanding of basic metabolism and its regulation. This in turn has led to new approaches inthe production of substances derived from multigene pathways such as antibiotics. Slowly, but inevitably, the once impenetrable barriers between species have been broken down. This rapidly developing field has also been accompanied abypublic debate about the ethics of genemanipulation and about environmental and safety aspects. It is not the intention of this bookto address thewhole field of gene technology, but rather to focus on one of the least contentious areas-gene V

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Preface

expression in recombinant microorganisms. Nevertheless, the chapter on regulatory and safety aspects will confront the environmental question and attempt to put into perspective the risksand benefits of this exciting technology now at our disposal. Scientists with international reputations have been recruited to compile up-to-date reviews on gene expression in recombinant microorganisms. Each chapter provides a specialist’s account of gene expression in one of the important classes of microorganism used to express both homologous and heterologous genes. Each chapter describes the state-of-theart for the expression system, provides examples of the expression of homologous and heterologous genes, documents the industrial and commercial exploitation of the system, and speculates on future prospects and developments. More precisely, the features discussed include host strain characteristics, selectable markers, protease-deficient strains, transformation systems, plasmid vectors, integrating vectors, copy number, stability, constitutiveand regulated promoters, activators, secretion signals, posttranslational modification, glycosylation, S -S bridges, and protein folding. Where appropriate, the authors describe aspects of metabolic regulation and fermentation process technology. The individual chapters are “validated”by extensive referenceto the published literature. The chapter on regulatory and safety aspects reviews current legislation in the United States, England, Europe, and elsewhere. The measures taken for large-scale operation are also addressed, including the evolution of good industrial large-scale practice (GILSP), as regulatory authorities seek to achieve a balance between safety requirements and regulatory excess. The gram-negative bacterium Escherichia colihas been thefront-runner in the commercial application of rDNA technology and there are highly developed fermentation processes operating on a large scale for wellestablished products. Joan Stader’s chapter describes how this leading position is based on the expanding knowledge about the basic biology of this organism. The availability of a range of regulated promoters provides numerous options for efficient gene expression. In particular, the system basedon the T7-RNA polymerase aispowerful tool for high-level expression of heterologous genes. However, this organism brings with it a number of problems. Among themare the difficulties of protein secretion across the dual membrane system of the gram-negative bacterium, the contamination of products with endotoxins,and the reluctance with which correctly folded proteins containing disulfide bridges are produced. The synthesis of fusion proteins is one way to overcome some of these

Preface

VI1

problems and can be combined with specific procedures for protein purification using affinity chromatography. The bacilli have a number of features that make these gram-positive bacteria potentially attractive hostsfor gene expression. There isa long history of successful industrial exploitation and the capacity to secrete large amountsof homologous proteins. In hischapter, Matti Sarvas describes the expression vectors based on host a-amylases and proteases. Most work has been done using Bacillus subtilis, but B. brevis also has promise as a host for the expression of heterologous genes. The major problems that have been confrontedare the degradation of secreted proteins by the host’s own exoproteases andthe apparently narrow rangeof heterologous proteinsthat can be secreted in reasonable yield. There appears to be an incompatibility with theBacillus secretory machinery in many instances.A case-by-case judgment is clearly called for since sometimes this host may indeed be the system of choice. Others have turned to the eukaryotes for solutions to problems encountered with prokaryotic host/vector systems. Thus the yeast Saccharomyces cerevisiae has been successfully exploited for the production of certain heterologous proteins. Hinnen and co-workers describe the expression system based on the indigenous 2-pm plasmid. Gene expression can be constitutive or inducible and there are examplesof the efficient secretion of proteins into the culture medium. The anticlotting protein hirudin is an example ofa heterologous proteinthat is well expressedand secreted. The host strain can also be tailored using molecular genetic procedures to eliminate proteasesthat would otherwise degrade the foreign protein. Experience has shown, however,that some proteinsare poorly secreted by yeast and glycosylation can present additional problems. The example of insulin-like growthfactor shows that there is still much to be learned about posttranslational events leadingto a correctly folded, biologically active product. The methylotrophic yeasts, represented by the genera Pichia and Hansenula, present an interesting alternative. Gerd Gellissenand colleagues describe the exploitation of the strongly inducible promoters associated with growth of these organisms on methanol. Here the use of a cheap substrate and the possibility of growing cultures to high cell densitiesare attractive features to the fermentation technologist. The constructs are stable by virtue of integration into the host genome, and in the case of Hansenula the foreign gene can be present as multiple copies. As with Saccharomyces, there are examplesof both intracellular and extracellular products.

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Other candidates among the eukaryotes are the filamentous fungi. Again, there is a wealth of experience in large-scale fermentationof these organisms, and the genus Aspergirlus could be a viable alternative to the other well-established host/vector systemsfor the production of heterologous proteins.Jaap Visser and his colleagues document thefungal expression system, based on the database of knowledge established for Aspergillus nidulans and Neurospora crassa and now extended to the industrially more interesting strains such as A . niger. The filamentous fungi are particularly interestingfor their abilityto secrete large amounts of homologous proteins. Practical illustrations of the potential of this class of organism are provided by the production of pectinases for use in the food and beverage industryand of glucose oxidase, which hasapplications in the production of gluconic acid, in construction of biosensors, and in diagnostics. The interest in recombinant DNA technology is not, however, restricted to the production of heterologous proteins. The interest in secondary metabolites, such as antibiotics, has a much longer history. The genus Streptomyces is a prolific producerof low-molecular-weight compounds of commercial interest. Here also, recombinant DNA technology has been exploited to improve strains, to create new hybrid molecules, and indeed to clone complete.biosynthetic pathways for certain compounds. The availability of vectors that replicate in more than one species has opened up the way to express individual genes or clusters of genes in the organism of choice. In Chapter 6, Richard Baltz has compiledan extensive review of gene expression and regulation in Streptomyces, which illustrates the excellent progress in understanding these organisms and also points to the work that still remains to be done. our intention to provide a state-of-the-art In compiling this book it was documentation of gene expression in the most important of classes recombinant microorganisms,to provide comprehensive reference to the literature, and to illustrate progress with concrete examples. We hope that this book will be of special interest to all those working in the fieldof gene expression, whether they are studying the regulation of primary or secondary metabolism, expressing homologous genes or heterologous gene products for the biopharmaceutical industry.It is also our intention that this book should be of general interest to those in the scientific community as a whole seeking a comprehensive description of the scientific and commercial application of microbial recombinant DNA technology. Alan Smith

Contents

Series Introduction Preface Contributors

1. Gene Expression in RecombinantEscherichia coli Joan Stader 2. Gene Expression in RecombinantBacillus Matti Sarvas Recombinant 3.Expression Gene Yeast in Albert Hinnen, Frank Baton, Bhabotosh Chaudhuri, Jutta Heim, Thomas Hottiger, Bernd Meyhack, and Gabriele Pohlig

4. Gene Expression in Methylotrophic Yeasts Gerd Gellissen, Cornelis P. Hollenberg, and Zbigniew A . Janowicz

iii V

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1 53

121

1%

ix

X

Contents

5. GeneExpressioninFilamentousFungi:Expression of Pectinases and Glucose Oxidase in Aspergillus niger Jaap Visser, Henk-Jan Bussink, and Cor Witteveen

241

6. Gene Expression Recombinant in

309

Streptomyces

Richard H. Baltz 7. RecombinantMicroorganisms:Safetyand Regulatory Aspects Anthony J. Taylor

383

Index

407

Contributors

Richard H. Baltz Lilly Research Laboratories, a Division of Eli Lilly and Company, Indianapolis, Indiana HenkJan Busink WageningenAgriculturalUniversity,Wageningen, The Netherlands Frank Buxton CIBA-GEIGY AG, Basel, Switzerland Bhabotosh Chaudhuri CIBA-GEIGYAG,Basel,Switzerland Gerd Gellissen Rhein Biotech GmbH, Dusseldorf, Germany

CIBA-GEIGYAG,Basel,Switzerland AlbertHinnen* CIBA-GEIGYAG,Basel,Switzerland

JuttaHeim

Cornelis P.Hollenberg Heinrich-Heine University, Diisseldorf, Germany

Thomas Hottiger CIBA-GEIGYAG,Basel,Switzerland Zbigniew A. Janowicz Rhein Biotech GmbH, Dusseldorf, Germany *Present qffiliution: Hans-Knlill-Institut fur Naturstoff-Forschung, Jena, Germany. XI

Contrlbutors

xll

BerndMeyhackCIBA-GEIGY

AG, Basel,Switzerland

Gabriele Pohlig CIBA-GEIGY AG, Basel,Switzerland Matti Sarvas National Public Health Institute, Helsinki, Finland Joan Stader University of Missouri -Kansas City, Kansas City, Missouri Anthony J. Taylor* Health and Safety Executive, London, England Jaap Visser WageningenAgriculturalUniversity,Wageningen,The Netherlands

Cor Witteveen Wageningen Agricultural University, Wageningen, The Netherlands

*Present qffiliation: Department of Health, London, England.

GENE EXPRESSION IN RECOMBINANT MICROORGANISMS

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l Gene Expression in Recombinant Escherichia coli Joan Stader University of Missouri-Kansas City, Kansas City, Missouri

1 INTRODUCTION Twenty years ago, the successful expression of heterologous proteins in Escherichia coli was largelya matter of luck. Atthat time, the focus was primarily on promoter strengthand how well the bacterium could tolerate the foreign gene or protein product. In many casesthe DNA sequence was not known, and genes were localized by restriction mapping based on a limited number of available enzymes. If gene expression failed to materialize, few options for troubleshooting were available because our understanding of recombinant gene expression was not sufficiently advanced. Much progress has been made since that time; and although some proteins are difficult to express in E. coli, a persistent researcher who takes a systematic approach is likely to succeed. It follows that the more we understand about the host organism in general, the better our chances of making it produce recombinant proteins. This is why E. coli is sucha good host-no organism is better understood in termsof genetics, physiology, or biochemistry. It is this wealth of information that has led to our present high level of sophistication in 1

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E. coli recombinant gene expression, and consequently, it is now possible to express genes from almost any organism inE. coli. The primary aimof this chapteris to summarize the molecular genetic principles that are germane to successful recombinant gene expression in E. coli and to present options for designing such a project. Many of the variables that affect gene expression itself are understood well enough that they can eitherbe controlled or counteracted. For instance, the diminishing effect of low plasmid copy number could be balanced by optimal promoter strengthand translation initiation signals. In some cases, a high plasmid copy number in combination with a weak promoter may yield the same results. It is likely that maximization of all aspectsof expression isunattainable because eventually one step will become limiting. Thus optimization may be achieved through a combination of choices that can be changedor substituted as the situation dictates. Our present ability to be flexiblein our approach to heterologous gene expression in E. coli means that the solution to the problem is still empirical but possible in almost all cases. In this chapter I do not recommand any specificapproach to gene expression because each gene presents unique challenges. However, since these challengesare not known at the outset, some general recommendations can be made with respectto the main issues of plasmid copy number, regulation of gene expression, promoter strength, and translation signals. It makes senseto take the simplestapproach at the beginningand make modifications along the way to optimize expressionof a particular gene. For example, use of a high-copy-number vector is advisable because highDNA yields makeit easier to perform in vitro DNA manipulations. Tight gene regulation is recommended because many genes are toxic when presenton a high copy plasmid. If the promoter is regulated by a repressor protein,it is important to have sufficientlevels of repressor available. The T7 phage promoter, which is currently very popular for gene expression, doesnot eliminate the necessity for repression but simply diverts the problem from direct repression of the gene of interest to repression of the T7 polymerase gene. Optimization oftranslation signals is dealt with most easilyby using vectors that allow the constructionof in-frame gene fusionsthat place the gene of interestat the 3’ end of the fusion. Of course, the gene fusionapproach would be unsuitable in cases where the hybrid proteinproduct is uselessfor the intended purpose.Beyond these general recommendations, other choices to be made depend on the project’s objective and required downstream operations, such as fermentation, protein purification, cleavage of fusion proteins, and re-

Expression Gene

Recombinant in fscherlchia coli

3

naturation. Finally, personal preference or researcher skills can be accommodated by alternative choices.A researcher who possesses greater skills in the area of DNA manipulation may be better off engineering an expression schemethat involves a more complicated cloning strategy to avoid protein purification problems later, while another researchermay be more comfortable with the reciprocal arrangement. In this chapter I discuss some of the pros and cons of the various approaches. Thus protein overexpression should be approached systematically, first addressing themajor issues of choosinga vector, then optimizing expression by making sequence changes or gene fusions,and finally, by experimenting with different host strains and growth conditions. The scaling up of expression for fermentation requires special considerations that need not be addressed in the early stages of development. These issues are not a primary focusof this chapterbut are addressed in instances where they seem relevant.

2 PLASMID VECTORS Most current examples of overexpression inE. coli utilize derivativesof pBR322. These plasmids carry a ColEI origin of replication and their copy numbers range from 20 to 700 per cell, depending on whether or not sequences requiredfor regulation of replication are present. There are good reasons for utilizing the pBR322 derivatives, such as a known DNA sequence, a proven track record, and knowledge of the details related to its mechanisms of replication, stability, mobilization,and amplification (1,2). Other repliconsthat differ from ColEI plasmids ainnumber of characteristics may alsobe useful for certain special situations (see Ref. 3 and citations therein);for example, derivatives of plasmid pSClOl are generally used when a low-copy-number vector is required. Most expression vectors carry a selectable marker and a strong promoter. Some vectors carry terminators, antiterminators, a repressor gene, translation signals, secretion signals, reporter genes, orthe potential for producing a hybrid proteinproduct. In Table1 are listed gene expression plasmid vectors in current use which are available commerciallyor can be obtained by request from the laboratories that developed them. Vectors designed primarilyfor general cloningand sequencing, such asthe pUC series, are also used for expression purposesbut are not listed. They can be used as an alternative to subcloning a gene into one of the expression vectors and customized through the of usecassettesthat carry someof the gene expression elements.

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Expression Gene

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Plasmid stabilityand copy number should be of concern an in expression system. Higher plasmid copy number will increase gene expression only if copy number is limiting the in first place. If the gene of interest is under the control of a repressor, an increase in copy number will be accompanied by an increased requirement for repressor molecules. If an antibiotic marker is present onthe plasmid, higher levels of the drugare required. In addition, high plasmid copy number places a greater demand on the physiologyof the host cell through its requirementfor factors involved in plasmid replication and maintenance. This situation may lead to slow growth of the host cells or outgrowth of cells that have a lower copy number.

2.1

Plasmid Stability

There are two types of plasmid stability: structural and segregational. Structural stability refers to the absence of insertions, deletions, and DNA rearrangements. Segregational stability refers to the retention of a plasmid in a population of growing cells. The stability of a plasmid should always be monitored by confirming the presence and size of the plasmid and assaying for production of the gene of interest prior to and during expression, especially during fermentation. Strains containing an overas frozen glycerol stocks and removed expression plasmid should be stored immediately priorto use -they shouldnot be usedif they have beenon a plate for several days. Depending onthe magnitude of the project, it is worthwhile to perform pilot experiments to determine growth conditions that will allow for optimal plasmid stability. In expression systems, structural stability is usually maintained through the avoidance of DNA sequences that contain intramolecular homologies and by utilizing areCA host. Nevertheless, insertion sequences present a potential problem, as these elements transpose randomly and independent of homologyor RecA function. Even though the frequency of transposition is low (about 1 in 108 cell divisions), insertional inactivation of a toxic gene confers a selective growth advantage when it arises in the population. There are no E. coli hosts lacking insertion sequences, but selection for rare insertional activation events can be avoided through the proper choice of transcription signals controlling expressionof the toxic gene (see below). Segregational stability is facilitated by par sequences present on some plasmid vectors that ensure plasmid inheritance to both daughter cells during cell division or by plasmid-encoded genes that confer a strong selective advantage in a given growth medium. Toxicity of resident genes can leadto segregational instability throughthe selective advantage con-

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Stader

ferred on plasmidless cells. Otherfactors, such as transcriptional activity of plasmid genes, growth conditions, selectable markers, and the genetic background of the host, strongly influence segregational stability. Any one of these factors could have a profound effect on protein yields. 2.1.1 SelectableMarkers In general, there are two ways to select for retention of overexpression plasmids in bacterialcultures: a suicidal mechanism or antibiotic resistance. In the suicidalapproach, the hostcell is missingan essential function that is supplied on the plasmid. For example, an amino acid auxotroph can be transformed with a plasmid that encodes a wild-type copy ofthe defective chromosomal gene. If the cells are grown on minimal medium lacking that amino acid,cells that do not inherit the plasmid will not survive. The advantagesof this approach are that it is inexpensiveand eliminates the possibility that the purified product will have antibiotic contamination. The disadvantages are that expression of the selected gene is not regulated as it normally is in the chromosome, and the daughtercells may obtain sufficient quantitiesof the gene product in the cytoplasm to survive for several generations in the absence of the plasmid. Antibiotic resistance is more commonly used because it allows for an extended host range and provides more flexibility in growth conditions; however, heterogeneous populations may arise when the antibiotic is inactivated or bound to the resistance geneproduct, lowering the effective concentrationsand allowing plasmidless cells to survive. The mode of action, mode of resistance, and working concentrations of the most commonly usedantibiotics are described in Ref. 3. 2.1.2 Transcription Levels and Plasmid Stability A decrease in the stability of plasmids canying nontoxic cloned genes under the control of strong promotersmay bedue to high-level transcription and replication interfering with one another. For instance, a comparison of high-level expression between a pBR322 derivative and a pUC derivative indicated that recombinant protein levels were much higher with the lower copy pBR322 derivative(4). The lower protein levels seen from the pUC derivativeseemed to be related to plasmid stability, and there was a strong correlation between high transcriptional activityand plasmid instability. 2. l.3 Plasmid Stability Under Various Growth Conditions Unfortunately, thereis no formula for increasing the stability of recombinant plasmids relative to growth conditions because every expression

Expression Gene

in Recomblnant Escherichia

coli

9

system is unique. In general, growth conditions that do not select for plasmid loss shouldbe used. Cells carrying plasmids require more energy than do cells that are plasmid-free(5); thus nutrient(6) or oxygen (7) starvation conditions should be avoided. It was reported recently that the presence of the bacteriophageX cos fragment made plasmid-containing cells as fit as plasmid-free cells when grown under starvation conditions (8). The reason for this phenomenon is not known, nor isit known if it will be generally applicable. Scaling-up operations from growth in shake flasksto continuous culture may result in a decrease in plasmid stability and(9) measures should be taken to monitor plasmid loss and devise schemes to avoid it. Sometimes this will involve altering the construct (7,9) or switching host strains. been reported (Q, in which the acquiThe use ofa "preadapted" strain has sition of one or more mutations during growth in a fermentor madeit more fit as a plasmid host, but the nature of these changes was not studied. Finally, systematic studies of plasmid vector stability have shown that the presence ofthe tet gene accelerates plasmid loss in the stationary phase (10) and there is a strong selection for mutations in tet (6,11), probably because the tet gene product is an inner membrane protein and E. coli has a limited tolerance for highly expressed inner membrane proteins.

2.2 Plasmid Inheritance and Copy Number Low-copy-number plasmids that are derivatives of pSClOl are usually present at around six to eight copies per cell (12). Inheritance of these plasmids is controlled by thepar locus, and they are stable even in the absence of a selectable marker (9,12,13). The par locus isa cis-acting site that is recognized by hostfactors to ensure proper partitioning, but it has no effect on copy number (12). Copy number in pSClOl is positively controlled by the plasmid-encoded replication protein, RepA (14), a protein whose expression is autoregulated (15). There is no partitioning function in pBR322 derivatives (ColEI replicons); thusthe maintenance of high-copy-number plasmids in a growing as described above. culture requiresthat they confer a selective advantage, Inheritance of multicopy plasmids is thought to be simplya matterof receiving a certain portion of plasmids residing in the cytoplasm,but this has never been proven. Copy number in ColEI plasmids is controlled by negative regulation of replication. Initiationof replication requires the binding of the plasmidencoded primer, RNAII, to the ori region (18,19). RNAII is first made of replication. Initiationof replication requires the binding of the plasmid-

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Stader

as a larger precursor which is processed by host RNaseH as an activation step prior to initiation of replication. Negative regulationof replication occurswhen another plasmid-encodedRNAmolecule,RNAI, hybridizes with RNAIIto prevent processing (20,21). Anadditional level of regulation comes from the plasmid-encoded rop (rorn)gene product, a repressor of transcription of RNAII (22). The levels of RNAIand Rop are regulated by plasmid copy number (gene dosage). Thus low number a of plasmid copies favors replication, and a high number of copies results in inhibitionof replication. For pBR322 derivatives, the normal number of copies present per cell is about 20 (2,3,23). This number can be increased five-to sevenfold through deletion of rop (U), and a further increase in copy number can be achieved through a point mutation immediately 5‘ to the coding region of RNAI (25). The pUC vectorsboth carry types of mutation, and the resulting copy number is about 600 (25).The effect on copy number of the latter mutation alone hasnot yet been reported. Several efforts have been madeto construct hybrid plasmids to obtain the high copy number of the pBR322 derivatives and the stable partitioning of the pSClOl (13,16,17). For instance, it wasfound that placing the pSClOl par locus on pBR322 increased plasmid stability 3- to 10-fold (16). Another example isthe “runaway replication” type of plasmid (13), in which a low-copy-number plasmid carrying the rep and par loci from synpSClOl is coresident with amutant ColEl replicon whose RNAII is thesized under XPLcontrol. These plasmids requirethe presence of the temperature-sensitive X repressor, cI857. At the nonpermissive temperature, plasmid replication occursand copy number will increase to about 200 copies per genomeafter 6 h. The temperature-sensitive replicon can also be used as a cassette, so that runaway replication canbe conferred on other plasmids.

2.3 ChromosomalExpressionVector A seriesof expression vectors has now been produced which are designed to reside in the chromosome (26). These vectors (MudMON) wereproduced for increased stability in the absenceof selection. The gene to be expressed is cloned into a plasmid vector that contains bacteriophage Mu sequences capable of transpositioninto the chromosome and can be maintained asa defective prophage.The gene of interest is placed under the control of the Mu middle promoter, which is efficiently repressed. In the presence of a temperature-sensitive repressor and a helper phage, replication of the vector and expression of the geneare both induced at

Expression Gene Recombinant in Escherichia

coli

11

42°C. Several heterologous proteins have been expressed with this system, and the levels seem to be dependent on the cloned gene itself. Because Mu has abroad host range, these vectors can alsoused be in other bacterial host strains, such as Serratia, Erwinia, Citrobacter, Pseudomonas, and Agrobacterium.

3 TRANSCRIPTIONSIGNALS With the exception of theT7 RNA polymerase-specific system, thepromoters utilized inE. coli expression vectorsare the u70 type because they are the most thoroughly understood. The bulk of routine gene expression in E. coli is handled by u70, and the promoters it recognizes are characteristically flexible in terms of levels of gene expression during changing growth conditions. The promoters are controlled at one or more levelsto respond to inducers and activators either specificto the operon in question or as part of global regulation systems. Many of the cis elements that participate in controllingu70 gene expression are a considerable distance away from the actual RNA polymerase binding site (27), and such remote control regions are usually not utilized in plasmid constructions. Therefore, regulation from a given promoter on a small, high-copy-number plasmid can be considerably different from that which takes place in a single copyon the chromosome. RNA polymerase holoenzyme recognizes a consensus DNA sequence centered at the - 35 and - 10 positions upstream of the start of transcription (+ 1). A close correspondence to the consensus sequence (Tathe rate-limiting ble 2) is required for efficient RNA polymerase binding, step in transcription initiation. Depending onthe operon, there are any number of cis- and transacting elements that serve to modify the ability of RNA polymerase to carry out a productive initiation (28). Once initiated, transcription usually proceedsto the termination site unless premature termination occurs. InE. coli, such premature termination occurs in cases of attenuation (29) or polarity (30-34). Both events depend on concomitant translationof the mRNA inwhich the ribosomestalls or dissociates from the message. When ribosomal stalling occurs, the message to the 3' side becomes void of ribosomes, allowing termination to occur. Attenuators are rho-independent terminators, which contain the canonical GC-rich region with dyad symmetry, capable of forming a hairpin structure, followed by a run of U residues at the 3' end. During translation, the ribosomes prevent the hairpin structure from forming; but if the ribosome stalls upstreamof this site,the hairpin structure will form and ter-

Expression Gene

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minate transcription. Stalling may occur at a codonfor which the aminoacyl-tRNA is limiting, such as conditions of starvation for a certain amino acid. E. coli has developed the ability to exploit this phenomenon inthe regulation of amino acid biosynthetic operons(29). Polarity sitesare rho-dependent terminatorsthat become active when the mRNA is void of translating ribosomes.When rho binding sites are exposed on the mRNA, the terminationfactor rho can bind and mediate the release of the transcript from a paused RNA polymerase elongation complex. Such RNA polymerase pause sites occur naturally within some transcripts (35). The rho recognition site is believedto be C-rich and includes a potential hairpin structure with a CAA loop and preceded byan rho recACCCCA consensus(36)based on in vitro binding studies, but ognition of such sites in vivo has not yet been reported. This type of premature transcription termination is beneficial to the organism because polycistronic messagescontain gene productsthat are often needed for a common pathway. Ifan upstream gene cannot be made because ofa frameshift or ambermutation, the downstream gene products wouldoften be useless. Transcription termination prevents the cell from expending the energy required to make these useless gene products. In the expression of genes in E. coli, premature termination can be avoided by examining the sequence of the gene for both rho-dependent and rho-independent termination sequences. If these sequences occur, they will not necessarily leadto premature termination; however, they can be modified by making conservative changesthe in DNA sequence. Alternatively, an ahtitermination mechanism can be employed through utilization of the bacteriophage X N-nut system. In the context of wild-typeX gene expression, theNgene is the promoter proximal gene under PLcontrol. After the initial induction of PL,a transcript of geneNis produced which ends at the terminator immediately followingthe gene (37). Upon translation of this message, N proteinacts on the nut (N-utilization) site (38)located downstreamof the promoter. When RNA polymerase passes this site, it acquires the ability to continue transcription through both rho-dependent and rho-independent terminators (38-40).This system is especially useful in cases where there is a long,untranslated 5’ region in the cloned DNA. It has been reported that use of the N-mediated antitermination system caused a three-to eightfold increase in protein production, and this system leadsto increased expression even in the absence of a terminator (41). Some expression vectors currently available provide the N gene and the nut site engineeredto prevent premature termination within the cloned sequence(42).

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3.1 Promoters The most highly utilized promoters for overexpression have two features in common: they are (a) efficient and (b) repressible. Some promoters can direct protein synthesisto near 50% of total cellular protein. This is accomplished by growing cultures to optimal density, performingthe required induction, and expressing the protein for several hours. Repressible promoters are superior to unregulated promoters because continuous expression of plasmid-encoded proteins generally puts the hostat a selective disadvantage ina population where some cells have lost the plascoli promoters used mid or acquired deletions and other mutations. E.The for expression of heterologous proteins are shown with the promoter consensus sequence in Table 2 and their salient features are presented below.

3.7.7 Plac In the chromosome, this promoteris negatively regulated by binding of the trans-acting repressor protein, LacI, to the operator site, lac0 (43, 44).There are three operators to which LacI can bind:one near the start of transcription, one at a remote site upstream,and one at a remote site downstream (27). Binding of the inducerto LacI causesan allosteric transformation of the repressor, leaving it unable to bind the operator (45). Lactose isthe natural inducer for this operon,but the gratuitous inducer isopropyl-6-D-thiogalactopyranoside (IPTG) is more powerful than lactose becauseit is not metabolized. In addition to inducer, expression from the lactose promoter requires cyclic AMP and the catabolite activator protein (CAP)(46,47). In the presence of the IPTG, cyclic AMP, and CAP,expression of IacZ is increased 1000-fold(48). The complex regulationof the lactose operon is greatly simplified in cloning vectors where onlyone operator is present, the commonly used lacUV5 promoter is independent of positive regulationCAP by (49), and the gratuitous inducer IPTG is employed to bring about sustained induction. Many of the popular cloning vectors in current use contain the lac promoter, followed by a multiple cloning site within the sequence encoding the LacZa peptide, allowingfor easy screeningfor insertions. The LacZa peptide consistsof 39 amino acidsof the amino terminusof LacZ @-galactosidase), which is capable of restoring enzymatic activity to a mutant LacZ missing amino acids 11 to 41 (the w-fragment) (50). The w-fragment is encoded by the IacZA MI5 allele in the chromosome of the host cell. In the absence of an insert, the plasmid will direct the expression of LaczOr, resulting in a blue colony on agar containing IPTG

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and the chromogenic substrate for &galactosidase, 5-bromo-4chloro3-indolyl-fl-~-galactopyranoside (XG). If foreign DNA has been inserted into the multiple cloning site, the coding regionfor L a c 2 is disrupted and the colony appears white. Thus genes cloned in these vectors can easily be expressed following induction withIPTG. Plac should carry To maintain plasmid stability, host strains carrying a lacZ allele that allows for constitutive productionof the lac repressor. The lacZq allele will result in the production of 10-fold more repressor .than that normally found inthe cell (51,52), but this level of repressor is normally not sufficient when high-copy-number plasmids are being used. Another allele, lacP, produces 100-fold more repressor (51-53) and is better suited for expression systems. Some cloning vectors carry IacZq on them and produce higherlevels of repressor asa result of the higher gene dosage. Such constructs confer more efficient repression of the promoter and also allowfor a broader host range.

3.7.2 Ptrp The product of the trpR gene negatively regulatesthe trp promoter (54). The aporepressorform is a TrpR dimer which is unable to bind tightlyto the operator (55). When complexed with L-tryptophan, the TrpR dimer becomes a repressor that tightly binds the operator and prevents the binding of RNA polymerase(54). Induction is brought about by tryptophan starvation conditions when TrpR is in the aporepressor form. Another level of regulation of the trp operon occurs at the attenuator, resulting in premature transcription termination inthe presence of tryptophan (29). The trpR gene is autogregulated, and it has been demonstrated that in cells growing in excess tryptophan, approximately 50 molecules of repressor are available to bind the operators for trp, aroH, and trpR (56). The relatively high levels of TrpR and its autoregulationare well suited to an overexpression system in which multiple copies of the operator are capable of titrating out the repressor. However, may one expectthat there is a limit to the capacityof the cell to regulate when the copy numberapproaches several hundred. Indeed, problems have been encountered after attempts to clone genes encoding toxic proteins undercontrol of the trp promoter. It is not clear whether these problems arise because repression of the promoter is insufficient orif the repressor has beentitrated out. Some of the trp expression vectors also employthe translation initiation region of the leader peptide, but the attenuator has been removed (57). Host cells bearing plasmids withthe trp promoter should carry the wild-type trpR allele. Induction is brought about by tryptophan starva-

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tion, which can be accomplished through leaky mutations inthe trp biosynthetic genes or through the use of the tryptophan analog indolyl-3acrylic acid (IAA) in combination with mildtryptophan starvation (57, 58). On a pBR322-derived plasmid with a copy number of20 to 30, the trp promoter is capable of directing the production of 20 to 30% of the total cellular protein after 3 h of induction (58). The decision to use the trp promoter should take into consideration the tryptophan content of the protein to be produced. It has been observed that proteins with a high tryptophan content are not expressed well under the conditions described(58). One possible explanation that is ribosome stalling as a result of tryptophan scarcity exposes polarity sequences inthe mRNA and leads to premature transcription termination. 3.7.3 Ptac and Ptrc These hybrid promoters possess the efficiency of the trp promoter and the regulation of the lacUV.5 promoter by combining the - 35 region of former withthe - 10 region of the latter (5339). The difference between the two hybrid promoters isthat tac contains 16 nucleotides betweenthe -35 and - 10 regions and trc contains 17 nucleotides (Table2). Full induction of tac with IPTG has been reportedto yield fivefold greater expression of cloned genes than the lac promoter (45). Vectors carrying these promoters are available, and cassettes that will convert a lac promoter to a tac promoter are also available.

3.7.4 Bacteriophage h Promoters PL and PR In their normal context within a lysogen, both of these promotersare repressed bythe bacteriophage X protein cI, which is requiredfor maintenance of lysogeny (60-162). Production of c1 from the Pm promoter is autoregulated, and a lysogen will normally produce 200 repressor molecules per.generation (63). Wild-type repressor activity is regulated by the host protein RecA. Upon DNA damage by chemical treatment or ultraviolet irradiation, RecA activates autoproteolysis the of X repressor, leaving the repressor unable to bind the operator (64). Under these condito bind RNA polymerase and initiate tions, bothPLand PRhave the ability transcription that will eventually lead to lysis. About 80 to 90% of the repressor molecules must be inactivated to bring about lysis (65,66). Expression systemsthat utilize X promoters requirethe presence of the repressor supplied eitheron a plasmid oron a prophage. Two induction schemes have been employed. One method involves the use of nalidixic acid (67), which will activate RecAand alter the cellular physiology through

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activation of the SOS response. The c1 cleavage reaction is quite slow, taking approximately 30 minutes to inactivate 200 repressor molecules (68). This approach presents two problems: (a) the promoter does not become rapidly activated,and (b) the dependence on RecA for induction is sometimes in conflict with the desireto use a reCA host. These problems can be circumventedby using the second induction scheme, which employs the temperature-sensitive c1857 allele (68). At the permissive temperature (30°C)the repressor binds the operator and promoter activity is shut off; at the nonpermissive temperature (42°C)the repressor is inactivated (independentof RecA) and the hostRNA polymerase beginsto transcribe PR-or P,controlled genes. Although heat induction is the promoters, problems arise most commonly used method of utilizing Xthe from the concomitant induction of the host heat shock response. The E. coli heat shock response induces the production of a variety of proteins, most notably cellular proteases, which could confound efforts to isolate recombinant proteins. Two possible ways of circumventing this problem are by using a heat shockmutant host strain [HtpR-, available as temperature-sensitive lethalmutations (69)] or pH induction. The latter approach has been described as being specific for the c1857 repressor and is independent of RecA (70). When the cl gene is present in a single copy and the X promoters are present in multiple copies, repression is not complete. Therefore, inclusion of the cl gene ona compatible plasmid oron the expression vector itself is beneficial, especially in cases where complete repression is essential. The X PLpromoter is about 8- to 10-fold more efficientthan PIac (41,71) and therefore about twice as efficientas Ptac. 3.1.5 Bacteriophage 7 7 Promoter Use of the T7 promoter system is described in detail elsewhere (72,73); a brief summary is provided here. Unlike the promoters described above, the bacteriophage T7 promoter requires the T7 RNA polymerase. Several advantages accompany this system: (a) transcription is completely selective-TT7 RNA polymerase will not recognize the host’s promoters; (b) Ti’RNA polymerase elongates mRNA chains five times faster than E. coli RNA polymerase,and the accumulation of message risesrapidly, approaching 100% of the cellular RNA synthesis (host mRNA synthesis can befurther suppressed bythe addition of rifampicin, an antibiotic to which the T7 RNA polymerase is refractory); and (3) expression of the cloned gene is completely activatable and a repressor is not required.

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However, the presence of T4 RNA polymerase can potentially negate this advantage if its synthesis isnot strictly controlled. Various approachesto repression of T7 RNA polymerase synthesis and activity have been developed, and one's choice dependson the toxicity of by induction the gene cloned.T7 RNA polymerase can be delivered either of a chromosomal copy of the geneor by infection with a specialized bacteriophage X transducing phage that carries the gene. In the former apapproach, the host strain is a lysogenthat carries a copy of the T7 RNA polymerase gene on the prophage, along with l a d . The T7 RNA polymerase gene is underthe control of the lacUV5 promoter and repressed until IPTG is added to the growth medium.The natural basal levelof activation of the lac promoter will result in a low level T7 of RNA polymerase synthesis, which may not be tolerated by the host cell if the cloned gene is highly toxic. If the cloned gene is highly toxic, low-level synthesis can be decreased effectively by the presence of T7 lysozymetheincell. T7 lysozyme isa bifunctional enzymethat hydrolyzes the bacterial cell wall (74)and inhibits T7 RNA polymerase (75). When presentat low levels in the cytoplasm, the lysozyme doesnot have accessto the cell walland will onlyact to inhibit the polymerase. The lysozyme gene is carried on a low-copy-numbercompatible plasmid in the host cell. Two versionsthat differ in thelevels of lysozymeproduced are available. For a slightly toxic cloned gene, low levels of lysozymeare sufficient; but highly toxic genes may require higher levels of lysozyme. An added feature of this system is that the presence of the lysozyme willfacilitate cell lysis duringthe protein isolation stage. It is best to use the lower levels of lysozyme if possible because the presence of lysozyme results in an initial lag in protein production following induction. Delivery of T7 RNA polymerase by phage infection is an approach developed for the expressionof genes that are toxic even in the presence of higher levels of lysozyme.By this approach, theT7 RNA polymerase gene is under the control of the strong XPLand PI promoters on the phage and is expressedas part of a lytic infection. The phage carries the temperaturesensitive repressor allelec2857 and Sm7, which prevents cell lysis (73,76). Under nonpermissive conditions (high temperature), this phage is unable RNA polyto enter the lysogenic state and will produce high levels of T7 merase, whichwill activate expression ofthe cloned gene. Synthesis of the cloned gene is so high that it interferes with phage development. The purpose of the amber mutation in theX S gene isto prevent cell lysis during expression. If this approach is utilized,the host strain should not be supF (76).

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3.7.6 OtherPromoters There are examples in the literature of the use of other promoters, some of which will be described below. Some of these promoters have been used only for certain types of expression systems, while others recently have been appliedto expression systems,and their utility isnot yet supported by a strong track record. Use of the PGpromoter from a naturally occurringPseudomonasputida plasmid has recently been described (77). The promoter is available as a cassette that can be utilized for expression inE. coli and other gramnegative bacteria. This promoter regulates the expression of genes required for the oxidation of salicylate to acetylaldehyde and pyruvate(78). Induction is brought about by the presenceof salicylate and the positive regulatory genenahR, which is also included on the cassette. Two phosphate-regulated promoters havebeen employed in expression systems. The first, PphoA, has been used in conjunction with the early coding regionof the phoA gene to construct a fusion betweenthe PhoA (alkaline phosphatase) signal sequenceand a heterologous protein (79). The purpose of these constructs is to target the recombinant protein to the secretion system (see below). The second, Pugp, has recently been reported to be effectivefor controlled overexpression of cloned genes (80). Its efficiency was shownto be about 80% of thatof P tac. Regulation of phosphate promotersis complicatedand still not fully understoodbut is known to involve multiple positive and negative trans-acting regulators (81). It has been reportedthat in addition to phosphate regulationPugp is under the global regulationof CAP, although it is unclear whetherthe latter is director mediated byanother regulator. The promoter is repressed in the presenceof glucose and phosphate, and induction occurs when glucose and phosphate are limiting. These conditionsare convenient and inexpensive and should be easilyadaptable to fermentation systems. 3.7.7 WhichPromoter Is Best? There isno standard answer to this question. Currently,the most widely used promoters are tac, trc, PR,PL,and W .The lac promoter is still used frequently, but the hybridtac and trc promoters offer the same regulation with higher expression levels. Since its development,the T7 system has become very popular and appears to have become the promoter of choice for high-level expression. Before choosingan expression system, it may be useful to examine the literature for expression of protein similar to the one you wish to express.

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3.2 Terminators Many examples inthe literature describe expression systemsthat utilize strong promoters withoutterminators behind the expressed gene. However, it has been shown that placement of a strong terminator at the 3' end of the gene results in greater stability of the plasmid (4) and greater expression of the recombinant gene(82). In the absence of a terminator, transcription can proceed into the plasmid, causing overexpression of other genes carried on the vector, potentially causing harm to the cell. For instance, overexpression ofthe tet gene can decrease the stability of the plasmid(6,ll). The presence of a terminator may be particularly crucial when cloning DNAfrom certain organisms(83). Several vectors currently availablecontain terminators on both sides of the multiple cloning site.

3.3 MessageStability mRNA molecules are commonly degraded in the 3'- 5' direction, and resistance of a message to exonuclease activity is conferredby a 3' stemof these strucloop structure(see Ref. 84 and citations therein). Thus some tures may actually play two roles; theyasact simple transcription terminators and they increase message stability. Message decay is mediated by both endonucleases and exonucleases. The exonucleases RNase I1 and polynucleotide phosphorylase (productsof the mb and pnp genes, respectively) are functionally redundant(85). Endonucleases that cleave mRNA include RNase I11 (the mc gene product) (Sa), RNase E (me/ams) (8789), and RNase K (88-90). The latter two enzymes play a general role in the chemical decay of bulk mRNA and there is currently a question toas whether they are separate, interdependent, or identical. RNase I11 has been shown to cleave RNA at specific recognition sites that contain double-stranded structures.This enzyme acts on various stable RNA speciesfrom bothE. coli and its bacteriophages and may play a role (91,92) or increasing turnover of cleaved mRNAs, in enhancing translation depending on the specific message(93-95). It has been proposedthat the degree of message stability is related directly to the efficiencyof translation initiation. The logic of this model is that closely packed ribosomes would make the message inaccessible to cellular RNases. Although the model may be true in some cases, an example in which it does not apply is the ompA mRNA. This message contains a structural element within its long 5' untranslated region (UTR) that

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confers a fivefold longer half-life on the adjacent coding region during periods of rapid growth. The stabilizing effect of the 5'UTR was found to be independent of translation initiation rate and adjacent coding region (96). Moreover, it has been determinedthat the ompA 5' UTR is a substrate of RNase K, which may control the rate of ompA turnover in vivo (90). The rolethat mRNA stability plays an in expression system can probe found or insignificant.If a toxic gene is being expressed and there is leaky expression froman uninduced promoter, enhancing stability of the mRNA may be lethal. Conversely, expression of a nontoxic genethat is poorly induced or carries weak translation signals may benefit from the addition of the ompA 5' UTR or expression in acell that carries an RNase defect. Indeed, useof an rnapnp double mutant defective for RNase I and polynucleotide phosphorylase has been shown to be effectivein improving the yield of recombinant porcine leukocyte interferon (97).If a gene product is not being expressed and message instability is suspected, turnover should be measured. Message stability canbe determined by measuring mRNA levels at various time intervals following the addition of rifampicin to the growth medium. These determinations should be done under the same growth conditions employedfor expression. 4 TRANSLATIONSIGNALS

It is at the level of translation that some of the most irksome problems arise in attemptingto overexpress heterologous proteins. The translation process is sufficiently different in prokaryotic and eukaryotic cells that it is not unusualto find sequences in the message that are incompatible with levels of gene expression in E. coli. At the root of the problem are two a message to be translated issues: (a) it is not fully understood what causes efficiently, and (b) optimization often requires changing nucleotides within the coding sequence of the gene of interest. Translation involves multiple components and steps (for current reviews see Refs. 98 and 99) and the resulting implicationis that each step is a potential target for regulation. Indeed, E. coli regulates the relative levels of geneproducts from a polycistronic message by alteringthe rate of translation through various mechanisms. Of all the components involved, mRNA isthe only highly variable one, and it is therefore the component that exerts the mostcontrol over translation rate. Optionsfor improving expressionat the translation level have been developedand will be presented later in this section.

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initiation

Initiation, elongation, and termination are all potentially rate-limiting steps intranslation, and of the three, the mostis knownabout regulation of expression at the stage of initiation. Multiple cellular components, including the 30s ribosomal subunit, the initiator tRNA, several initiation factors, and messenger RNAcontrol the fidelity and efficiency of the initiation process. It is essentialthat the mRNA meet strict criteria ensuring that translation always begins at the proper AUG codon and not elsewhere. Comparison ofthe initiation regionof a number of E. coli genes (100-104)as well as mutational analysis (105-113)has made it possible to determine someof the “rules” involved inthe process that aredescribed below. Still, much remainsto be elucidated since thereare numerousexamples of messages that do not conform to our composite pictureof what determines a good initiation site and yet are efficiently translated(98,99, 114). There are also sequences (“nongenes”) that conform to the rules that do not act as initiation sites (103,115,116).It could be that our inability to predict translation efficiency reliably isa reflection of our inability to predict mRNA secondary structure accurately.

4.1.1 RibosomeBindingSite (RBS) The ribosome binding site (RBS) isfunctionally defined as the portion of mRNA that is protected from RNase digestion by ribosomes that have bound but not initiated translation (117). This region consists of about 30 to 35 nucleotides extending from about -20 to + 13 of the message (+ 1 being the first nucleotide of the initiation codon). The individual the , initiation components are the Shine-Dalgarno (SD) sequence (la) codon (105),the spacing betweenthe SD sequenceand the initiation codon (103,114),the second codon(102),and other elementsof the primary sequence that are notwell understood. Occlusionof the SD sequenceand/ or thestart codon hasa profound effect on translation efficiency (1 18-122).

4.l. 2 Shine-Dalgarno Sequence It is thought that the purpose of the SD sequence isto allow for proper alignment of the ribosome with the initiation codon (123). The SD sequence isa purine-rich sequence positioned5 to 12 nucleotides upstream of the initiation codonthat is complementary to nucleotides at the 3’end of the 16s rRNA (104): rRNA: 3’AUUCCUCCACUs‘ mRNA: S’UAAGGAGGUGA3’

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The length of theSD sequence on the message varies, but the average is five nucleotidesand the majorityof E. coli messages studied so far carry the central sequences (underlined above) (103,115). Hybridization between the twoRNAs has been demonstrated biochemically (124) and genetically (125,126). There are conflicting opinions on the effect of longer SD sequences on the efficiencyof translation (99,123). This issue has been addressed in several studies, and the answer is not clear-cut. For example, a single base change that resulted inGAAG to GGAG increases synthesis 10-fold (127), while increasing complementarity to 13 nucleotides decreased synthesis by a factor of 2 (128). Context probably plays a role in regulating these expression levels. Clearly, if therea isnegative effect from a longer sequence, it is not large. 4.7.3 Spacer Region Between the SD Sequence and the Initiation Codon The distance between the SD and the initiation codon ranges from 5 to 13 nucleotides (99) and depends on the SD sequence and perhaps other elements inthe mRNA. The variability in the distance implies a precise distance between the region of complementarity inthe 16s RNA and the site of the initiator tRNA bound to the ribosome. The nucleotide sequence 1 triplet, affects in the spacer region, specifically the three bases in -the the initiation rate up to 20-fold (112)the most favorable beingUAU and CUU and the least favorable UUC, UCA, and AGG. 4.7.4 InitiationCodon The most commonly used initiation codonis AUG, but occasionally,UUG and GUG are employed in E. coli (105). 4.7.5 SecondCodon A systematic study revealed that (a) the second-position codon choice deE. in coli, and (b) there is a wide viates strongly from overall codon choice variation in translation efficiency depending on the choice of codon in the second position(102). It was demonstrated that the most commonly used second codon, AAA, is also themost efficiently translated, and these data are consistent with another study showing a strong bias towardAs in the more efficient ribosome binding sites (129). There was considerable variation among synonymous codons, suggesting that the nucleotide sequence is responsible for the effect. This study examined the effect of changing the second codon within given a context (thelacZ gene) and does not necessarily apply to other genes. For example, changing the codon

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from GAUto AAA in one context increased protein synthesis 10- to 15fold (102), while in another,an increase of onlytwo- to threefold was found (4). It is clear that for a given amino acid in the second position, some codons will be better choices than others.

4.2 Elongation In contrast to initiation, there seems to be no effect from mRNA secondary structure on elongation. Ribosome stalling during elongation can occur if stretches of rare codons appear in the message or if stretches of a single amino acid are required and the cell is starved for that amino acid,as in the case of attenuation. Ribosome stalling resultingfrom the appearance in the coding region of a SD sequence isalso believed to play a role in the frameshiftingthat occurs duringtranslation of release factor 2 (RF2) (130).

4.3 Termination

Unfortunately, little is known about the existence of regulatory mechanisms actingon translation termination. It is conceivablethat even a slight stalling at the stop codon could affect proteinlevels dramatically by caussubsequent ribosomes to back up. Whether this situation ever arises in normal translated messages has not been studied to my knowledge; but given that it isa multicomponent mechanism, it would be difficult to imagine that E. coli has not expolited its regulatory potential. 4.4 Optimization of TranslationEfficiency

Designing optimal translation efficiency into an expression system has been described previously(1 14). Briefly, translation initiationcan be optimized if consideration is given to the following factors: 1. mRNA sequences that have intramolecular structures involving the SD or the initiation codon should be avoided. 2. A long (five to eight nucleotides) SD is favorable for translation. 3. The spacing between the SD and the initiation codon is important

but depends on the length and sequence of the SD. As a result of the precise distance between the 3' end of the 16s rRNA and the binding sitefor the initiator tRNA inthe ribosome, longer spacing is more tolerable than shorter spacing. 4. AUG is most favorable as an initiation codon.

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5. The gene of interest should have >25 nucleotides 5’ to the initiation codon.If this region is extensive, translation termination codons should be present upstream of the ribosome binding site(RBS). 6. Second codon choice should be optimized. Rules for nucleotide choice downstream of the initiator codon are difficult to define and may be somewhat irrelevant in designing an expression system due to the constraint imposedby specific amino acid requirements the in protein product and the difference in context. For these sequences, optimization is restricted to silent codon changes.

Several approaches have been developed for optimization of translation initiation. They include (a) cloning the gene directlyinto a vectorthat has a strong promoter followedby a SD sequence and initiation codon, (b) constructing an in-frame gene fusion that contains the 5‘ region of a gene knownto have goodtranslation signals fusedto the gene of interest, (c) site-directed or random mutagenesis of the early coding region followed by screening for high-level synthesis of the gene product, and (d) placement of a short cistron upstream of the gene of interest to increase the chances of reinitiation at the downstream RBS.

4.5 Direct Cloning into a Translation Vector Several families of vectors have been developed that contain a strong promoter followed bytranslation signals and carry a unique restriction site at the start codon. Use of such a vector is aidedby the fact that a large proportion of eukaryotic genes contain an NcoI site(C’CKGG) at their start codon (131). Other restriction sitesthat carry the ATG sequence are NdeI, BspHI, and Aj7III. For genesthat do not carry one of these sites, one can be introduced by site-directed or PCR mutagenesis. Table 1 indicates the presence or absence of translation signals within expression vectors. Vectorsthat utilize the direct cloningapproach generally havea + / - ” under the “hybrid protein” heading because theyoften provide the option of cloning into one of the ATG-containing restriction sites,or cloning into a downstream site that will result in one or more extra amino acids incorporated into the amino terminus of the protein. The direct cloningapproach does not take into account the role of the early coding region in translation initiation. Therefore, if expression is not achievedby direct cloning,the problem may be circumvented by one of the following schemes.

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4.6 In-frameGeneFusions

These constructionsare a useful way of avoiding problems withtranslation initiation and also provide other advantages (see Section 6). First, cloning is simple. Several families of fusion vectors are available that carry unique restriction sites in all three reading frames, eliminating the need for nuclease treatmentor site-directed mutagenesis. These vectors appear in Table 1with a “+” under the “hybrid protein” heading. Second, the translation initiation signals have been optimized. Third, the amino-terminal moiety may provide a simple assayfor expression, a means of affinity purification of the hybrid protein, protection against proteolysis, or competence for export out of the cytoplasm. The major disadvantageto using a fusion protein arises in cases where the hybrid protein is not useful as such and requires precise processing and purificationto yield an authentic protein product. However, methods for precise processing of fusion proteinsare being aggressively pursued and thereare some very well-designed schemes available that employ linker regions carrying protease recognition sites between the amino and carboxy terminalparts of the hybrid protein. 4.7 Site-DirectedandRandomMutagenesis If expression levelsare too low and production of a fusion proteinis not desirable, the early coding region can be mutagenized. Sometimes the problem may be an obvious secondarystructure causing blockageof all or part of the RBS that can be altered by site-directed mutagenesis. If the problem is not obvious, random “cassette” mutagenesis may be the method of choice. This approach involves the synthesisof complementary oligonucleotides in which random incorporation of the nucleotide in the wobble position of each codon is carried out. The heterogeneous mixture of nucleotides is then clonedinto the gene, and transformants are screened for production of the recombinant protein. Thisapproach has been utilized effectively in the expression of human glutathione reductase(132).

4.8 Two-CistronTranslationSystem

This approach (133) is applicable in cases where secondary structure within the mRNA occludes theRBS. The rationaleis based on numerous instances in E. coli where “translational coupling” (134-136) occurs. Occasionally, one of the downstream genes in a polycistronic message will have occlusion of the RBS due to mRNA secondary structure, resulting in the in-

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ability of the ribosome to bind and initiate translation. Ribosomes that are translating the upstream gene are capable of destroying the secondary structure in the downstream RBS if the stop codon of the upstream gene is closeto the start of the downstream gene.It is thought that such an arrangement leadsto “reinitiation” at the downstream gene immediately following termination of the upstream gene. The two-cistron system was shown to work in one casefor a gene that was transcribed and not translated, but it did not improve translation of a gene that was already efficiently translated (133). A two-cistron system was used in an attempt to express human glutathione reductase (132). In this case expression wentfrom undetectable to low levels but did not approach the levels expressed following cassette mutagenesis. In two other cases, such a system wasreported to yield high levels of recombinant protein(137,138). The applicabilityof this approach would seemto be limited to those cases where there isother no acceptable way of removing secondary structure at the RBS.

5 POSTTRANSLATIONALEVENTS Following synthesis ofa recombinant protein in E. coli a number of events may occur, some desirable, some benign, and some undesirable. These can be placed into two general categories, nonreversible, such as covalent modifications and proteolysis, and reversible, such as misfolding and aggregation. Onthe other side of the issue, thereare some posttranslational modifications that do not occur inE. coli that are part of processingand assembly operations that many eukaryotic proteins normally undergo in nature. Fortunately, in some cases the lack of these normal modifications does not diminish the biological activity of recombinant proteins. For example, recombinant proteins produced inE. coli do not become glycosylated, yet the biological activity of these proteins in some cases is the same or higher than that of the natural product (130). Moreover, it has been reported that the recombinant proteins have a longer half-life in the blood system (140,141). For this reason, recombinant proteins may be superior for some pharmaceutical purposes. 5.1 Nonreversible Events

This group of modifications includes reactions such as removal of the amino terminal fMet residue and proteolytic cleavage reactions. Other posttranslational modifications, such as methylation, acetylation, and

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fatty acylation, seem to be highly specificfor certain E. coli proteins and will not be addressed further. 5.1.1 Removal of N-Formylmethionine It is believed that the nascent polypeptide is first deformylatedand then the Met residue is removed by a Met-specific aminopeptidase. This view is based on the identificationof formylase activityfound in E. coli extracts and the lack offMet activity (142). However, the Met-specific aminopeptidase has not yet been found, and a full understanding of the process will have to await the identification and purification of this activity. The nature of the second amino acidaisdetermining factor in whether or not the fMet residue is removed from a polypeptide in both eukaryotes and prokaryotes(143). Those amino acidsthat favor removalof fMet are universally conserved in various organisms,and they are Ala, Ser, Gly, Pro, Thr, and Val. Those that disfavor removalare Arg, Asn, Asp, Gln, Glu, Ile, Leu, Lys,and Met. In some cases of recombinant protein production inE. coli, only a proportion of the protein product has had the fMet removed (144). This situation may result from overloading the cellular processing machinery by such a high levelof protein synthesis. 5.1.2 ProteolyticReactions E. coli has may proteases,both specific and nonspecific (for current reviews, see Ref.145 and 146). Specific proteolysis occurs routinely by signal and by signal peptidase peptidase Ias part of the general export pathway 11, in combination withother covalent modifications, in the localization of lipoproteins. In responseto various environmental conditions, specific proteolysis also plays a role, such that as medited by RecA as part of the SOS pathway. Numerous nonspecific proteases have been identified in E. coli and more are likely to be discovered in the future. The profile of nonspecific degradation changes with growth conditions. For instance, abnormal proteins that result from misincorporation of amino acids or truncation are routinely degraded under all growth conditions; (145). but under starvation conditions, normal proteins are also degraded Under most starvation conditions, proteolysis increases in the early stages but declines as starvation persists. Proteolysisis particularly high under nitrogen starvation. At least one protease, Clp, has been shownto have its highest activity during late exponential and early stationary growth phases (147). The most highly characterized E. coli protease is Lon, which acts on abnormal proteins but also cleaves normal proteins inthe regulation of

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capsular synthesis and cellular stress responses. For example,as part of the SOS response, the cell division inhibitor SulA is produced. Following DNA repair, it is necessary for the cell to rid itself of SulAto allow cell SulA through division to resume. The role of Lon in this case to inactivate is proteolysis (148). If Lon isabsent, SulA continuesto inhibit cell division, and eventually the cell becomes filamentous and dies. Thus strains that do not produce Lon are sensitive to treatments that induce the SOS response. Lon is also one of the proteases regulated by the heat shock response (149). This global control circuit is regulated by the alternate a-factor, HtpR (69,150-152), which is required at all temperatures,but higher levels are required at elevated temperatures. Recently, another protease, DegP/ HtrA (153,154), has also been identified as a heat-shock protease (155, 156). Some protease activity is confined to particular compartments. For instance, the periplasm contains DegP, protease I11 (157) [(product of the ptr gene (158,159)l and possibly theproduct of the sohB gene (160). The latter is homologousto protease IV,an enzyme that degrades cleaved signal sequences in the inner membrane (161). Protease V resides in both the inner and outer membranes ofE. coli and has specificityfor L-glycine and L-phenylalanine derivatives(162,163). The outer membrane contains protease VI, a serine protease (164), and protease VI1 (OmpT), which has been shown to have specificityfor paired basic residues(165). Even if a recombinant proteinis retained in the cytoplasm during synthesis, these extracytoplasmic proteases can create problems during the purification stages following cell breakage. The use of protease-deficient strains for the expression of recombinant proteins inE. coli has been described(166). Strains that carry nullmutations of Ion are availableand can be usedas hosts, or the mutation can easily be introduced into any strain by P1 transduction. Lon mutants have pleiotropic phenotypes stemming from the regulatory functions described above. One phenotype is mucoidy, resulting from unregulated capsule synthesis (167). This phenotype can create difficulties in harvesting cells from liquid culture, isolating colonies, and performing phage infections. Mucoidy can be controlledthrough careful choiceof media or by using secondary mutations that map within genes necessary for capsule synthesis (166). Another phenotype of Lon- strains is sensitivity to DNAdamaging agents stemming from the cell’s failure to degrade SulA. Several concerns arise from this situation. One is that a Ion- mutation shouldnot be used in combination withdam- mutations becauseit results in lethal

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induction of the SOS response, and it follows that ion- strains should not be used in combination with promoters inducibleby nalidixic acid. Another problem is the Zon- strains cannot be grown in media containing yeast extract (e.g., LB). These phenotypes can be avoidedby using S U M mutants (166). Mutations inhtpR have been used in expression strains. The advantage to inactivation of htpR is that it results in decreased synthesis of several cellular proteases, including Lon. The HtpR defect is most commonly employed by usingan amber mutation inhtpR in combination with a temperature-sensitive amber suppressor(69,168,169). At the permissive temperature the cells produce HtpR, and at the nonpermissive temperature HtpR will no longerbe produced. With these strains, cell lysis occurs soon after the temperature shift. Lon protease is not completely absent in htpR mutants, and therefore Zon htpR, double mutants can be used. Finally, factors relatedto the induction scheme may diminish or exacerbate the problem of proteolysisof recombinant proteins. Thermal induction of promoters in HtpR+ strains will increase proteolysis in all compartments of the cell. In addition, raising the temperature may lead to aggregation of the recombinant protein, which could either make it inof proaccessible to proteases (170,171) or it could increase the likelihood teolysis (172). Rapid, high-level expression of proteins sometimes leads to aggregation even at lower temperatures(173,174) and may simply overof the recombinant proteininto the load the cellular proteases. Secretion periplasm will allow it to escape cytoplasmic proteasesbut will expose it to periplasmic or outer membrane proteases. It should be pointed out that proteolysis is only one potential cause of low expression levels. Before any attempt is made to eliminate cellular proteases, a pulse-chase study should be doneon the proteinof interest to see if proteolysisis actually a problem.

5.2 ReversibleEvents The most common reversible reactions encountered in expression systems are the formation of insoluble aggregates (inclusion bodies) or the misfolding of proteins. Misfolding maybe avoided if the protein can be successfully secreted into the periplasm, especially if the protein contains intramolecular disulfide bonds(175-177). For these proteins, the E. coli enzymes peptidyl-prolyl cidtrans isomerase and protein disulfide isomerase may be limiting. This problem has been dealt with successfully

Expression Gene

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through the use of the pBR322-compatible plasmid pASK61, whichcarries the dsbA/pdi and ppi genes (178). Host cellsthat contain both pASK61 and the pBR322-based expression plasmidsare grown inthe presence of the thiol-reagent N-acetyl-L-cysteine to provide optimal conditions for homogeneity of disulfide bondformation. Even interchain disulfide bonds have been folded correctlythe in case of the antigen-binding fragment (Fab) of antibody (175,179). This feat was accomplished by cloning the genes for the two polypeptide chains together so that a dicistronic message was produced. Presumably, the polypeptides are synthesized and secreted with close proximity to one another, allowing them to assemble correctly. Other examples of simultaneous expression of interacting polypeptides improving stability through proper folding are the of cAMPdependent protein kinase (PKA) regulatory and catalytic subunits (172) and human hemoglobin( 180). Secretion of the recombinant protein in the periplasm doesnot always eliminate aggregation (173,174,181), but the addition of nonmetabolizable sugars to the growth medium has been shown to decrease periplasmic aggregation (173,182). In some cases, aggregationaisby-product of heat induction (170),171)or a high synthesis rate (73,174); however, many examples are also found where thesefactors do not leadto aggregation or misfolding. Aggregation can be beneficial in terms of making the protein less susceptibleto proteolysis and easier to purify from the bulk ofE. coli proteins. 5.3 InclusionBodies If a protein formsan inclusion body, sometimes it is best to isolate it as such and then solubilize it. For proteins secreted into the periplasm, a simple method involves harvesting the cell, resuspending them in chloro(183). The simplest form, and recovering the protein from the supernatant procedure for the isolationof inclusion bodies associated with other cell fractions involves harvesting the cells and resuspending the pellet inTris/ EDTA/lysozyme/Triton X-100 to solubilize the cell envelope followed by sonication to reduce the viscosity caused bythe presence of DNA.The addition of DNase is typically avoided because the enzyme requires magnesium, which may also activate proteases. Centrifugation of this preparation will pellet the inclusion bodies. Solubilization of inclusion bodies is typically done with 8 M urea or 6M guanidine-HCI, and refolding requires dilution or dialysis in a buffer suitable for the particular protein

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involved. Thereare several sources in the literature describing procedures for protein solubilityand folding (184-186), and commercial suppliers of expression vectors often provide detailed protocols for the isolation of recombinant proteins. Examplesof biologically active recombinant proteins that have been isolated from inclusion bodies include mouse interleukin-6 (IL-6) (187), human IL-6 (188), human interleukin-5 (IL-5) (189), and horseradish peroxidaseC (190). 6 SPECIAL DESIGNS: FUSION PROTEINS

In this sectionwe deal with some special designs for expression of recombinant fusion proteinsthat take into consideration many of the individual concerns described in previous sections (for a recent review, see Ref. 191). Fusion proteins constructedfor expression systems usually contain an E. coli sequence at the amino terminusand the protein of interest at the carboxy terminus. This class also includes proteins engineeredfor secretion (even if the finalproduct is not a fusionprotein, the primary translation product is). Some of the potential benefits of expressing heterologous proteins as hybrid proteins are: (a) The presence of an efficiently translated E coli protein moietyat the amino terminus eliminates concerns about construc(b) in some cases, especially with tion of an optimal ribosome binding site; small peptides, a fusion protein will be more stablethan a nonfused protein (192); (c) purification of the hybrid protein by affinity chromatography is possible even if there is no ligand available for the heterologous protein of interest; (d) a variety of cleavage options are available, including chemical cleavage,or there are specially engineered linkersthat contain specific protease recognition sites so that cleavage can be carried out to yield an authentic protein; and (e) if the amino terminus carries a signal sequence, the fusion protein may be secretedinto the periplasm successfully, greatly simplifying purification and providing a recombinant protein withan authentic amino terminus (for review a of secreting heterologous proteins inE coli, see Ref. 193). The potential disadvantages of fusion proteins are: (a) Except in the case of secretion, the amino terminusof the cleavageproduct may have extra amino acids attached; (b) there may beadditional sites on theprotein of interest that are susceptible to cleavage by the treatment used to remove the N-terminal moiety; and (c) conditions usedfor chemical cleavage may damage the protein of interest.

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6.1 StaphylococcalProtein

coli

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A Fusions

Protein A from Staphylococcus a u r w binds specificallyto the Fc portion by IgG Sepharose of IgG and therefore proteinA fusions can be isolated chromatography. The latest versions of the pRIT series of vectors (Table l), pRIT20 and pRIT30, are available in two types for expression of protein A fusion inE. coli. The pRIT20 series is usedfor secreted fusion proteins and is recommendedfor proteins that are normally secreted in their natural hostcells. The pRIT30 series is designed for use with proteinsthat are not secreted in their natural host. Both typesof vectors contain linkers for all three reading frames,and these linkers encode the recognition sequence for either enteropeptidase, collagenase,or factor X,proteases. Details of the constructionand use of these vectors have been described (194). A specialized expression system called EcoSec has been developed (195). This system is designed for the expression of peptide hormones in soluble form and has been used successfullyfor insulin-like growthfactors I and 11. The EcoSec system utilizes plasmid vector pEZZ18 (Table 1).

6.2 p-GalactosidaseFusions Both amino- and carboxy-terminal fusion of @-galactosidase have been used for years for a wide variety of purposes.In general, @-galactosidase fusion proteins are among the largest polypeptides produced inE. coli and therefore,gel filtration chromatography canbe utilized. Anti-@galactosidase affinity resinsare also commercially available. Two recent applications of @-galactosidase fusion technologyfor engineered expression systems are the pSS20 (196) and the PAX (197) series of plasmids. There has also been a series of low-copy-number vectors developed for the production of @-galactosidase fusions (198).

6.3 GlutathioneS-TransferaseFusions Fusions to gluthathione S-transferase (GST) can be purified on Glutathione Sepharose 4B. Two fusion vectors are commercially available, pGEX2T and pGEX-3X (Tablel), which carrythe recognition sitesfor thrombin and factorX,,respectively. Another vector,PGEX-~T,encodes ahistidine hexapeptide linked to GST for the expression of fusion proteins that can be purifiedon metal chelation resins (199).

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6.4 Maltose-BindingProteinFusions Vectors designedfor the productionof fusions to the E. coli maltose-binding protein, with or without a signal sequence, can be purified by affinity chromatography on cross-linked amylose (200). Vectors encoding the recognition sequencefor factor X,are also available (201).

6.5 SecretionSystems In addition to the systems described abovethat secrete fusion proteins, plasmid vectors that carry the E. coli signal sequencesfrom phoA (181, 200)or ompA (202,203) have been constructed. Successful secretion using these vectors is more likely if the protein is normally secreted in its natural cell type. All of the vectors currently available target the protein to the general export pathway inE. coli, even though there are several specialized secretion systemsthat also occur in this organism. Success has been good in getting proteins exportedto the periplasm, but there have been no major successes in secreting recombinant proteinsinto the medium. Sometimes large amounts of proteins are indeed found in the medium, but they probably get thereby leakage.

7 CONCLUDING REMARKS The expression of heterologous genes in E. coli has benefitted greatly from our expanding knowledgeabout the basic biology of this organism. Our deeper understanding of the steps in transcription has to the led availability of a number of promoters whose activity can be controlled very effectively using repressors and inducers. Use of the l7 promoter presents a new twist-an activatable promoter. Hopefully, the success of this system will encourage more experimentation withother activatable promoters, such as those that use alternate a-factors. Translation has been more difficultto understand in general, because distinguishing the basisfor codon choice (nucleotideor amino acid) confounds algorithmic analysis. Unlike transcription, the process relies on nucleotide sequences within the coding region theofgene, an area the researcher is not always at liberty to change. The choice for optimization of translation initiation always depends greatly on the intended use of the recombinant protein. Controlling posttranslational events isan area where much work needs to be doneat the basic level.To achieve more cost-effective recombinant protein production fromE. coli, it will be important to learn as much as

Expression Gene

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CO//

35

we can about proteolysis and locabation of proteins. The genetic approach willand prowill be helpful in achieving greater understanding in this area vide useful host strains. The physiology of recombinant E. coli is another area that should be studied further so that we can continue to improve our chances of designing host-vector systems that are able to thrive in continuous culture.

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13. Mashko SV, Mochulsky AV, Kotenko SV, Lebedeva MI, Lapidus AL, Mochulskaya NA, IzotovaLS, Veiko VP, Vinesky VP, Ketlinsky SA, Debabov VG.Use of a dual-origin temperature-controlled amplifiable replicon for optimization of human interleukin-lp synthesis in Escherichia coli. Gene 1991; 97:259-266. 14. Vocke C, BastiaD. DNA-potein interaction at theorigin of DNA replication of the plasmid pSC101. Cell 1983; 35:495-502. 15. Linder PG, Churchward G, Guisizn X, Yi-Yi Y, Car0 L. An essential replication gene, repA, of plasmid pSClOl is autoregulated. J Mol Biol 1985; 181: 383-393. 16. Skogman G, Nilsson J, Gustafsson P.The use of apartition locus to increase stability of tryptophan-operon-bearing plasmids in Escherichia coli. Gene 1983; 23:105-115. 17. Zurita M, Bolivar F, Soberdn X. Construction andcharacterization of new cloning vehicles. VII. Construction ofplasmid pBR327par, a completely sequenced, stable derivariveof pBR327 containing thepar locus of pSC101. Gene 1984; 28~119-122. 18. Tomizawa J-I, Itoh T. The importance of RNA secondary structure inColEl primer formation. Cell 1982; 31575-583. 19. Davison J. Mechanism of control of DNA replication and incompatibility in ColEl-type plasmids: A review. Gene 1984; 28:l-15. U). Tomizawa J-I. Control of ColEl plasmid replication:The process of binding of RNA I to the primer transcript. Cell 1984; 38:861-870. 21. Tomizawa J-I. Control of ColEl plasmid replication: Initial interaction of RNA I and the primer transcript is reversible. Cell 1985; 40527-535. 22. Cesareni G, Muesing MA, Polisky B. Control of ColEl DNA replication: The rop gene product negatively affects transcription from thereplication primer promoter. Proc Natl Acad Sci USA 1982; 79:6313-6317. 23. Covarrubias L, Cervantes L, Covarrubias A, Soberdn X, Vichido 1,Blanco A, Kupersztoch-Portnoy YM, Bolivar F. Construction andcharacterization of new cloning vehicles. V. Mobilization and coding properties of pBR322 and several deletion derivatives includingpBR327 and pBR328. Gene 1981; 13~25-35. 2 4 . Twigg AJ, Sheratt D. Trans-complementable copy numbermutants of plasmid ColEl. Nature 1980; 283:216-218. 25. Chambers SP, Prior SE, Barstow DA, Minton NP. The pMTLnic-cloning vectors, I. ImprovepUC polylinker regionsto facilitate the use of sonicated DNA for nucleotide sequencing. Gene 1988; 68:139-149. 26. Weinberg RA, DeCiechi PA, Obukowicz M. A chromosomal expressionvector forEscherichia coli basedon the bacteriophage Mu. Gene1993; 126:2533, 27. Collado-Vides J, Magasanik B, Gralla JD. Control site location and transcriptional regulation in Escherichia coli. Microbiol Rev 1991; 55:371-394.

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77. Yen K-M. Construction of cloning cartridgesfor development of expression vectors in gram-negative bacteria. J Bacteriol 1991; 1735328-533s. 78. Yen K-M, Serdar CM. Genetics of naphthalene catabolism in pseudomonads. Crit Rev Microbiol 1988; 15:247-268. 79. Craig SP 111, Yuan L, Kuntz DA, McKerrow JH, Wang CC. High level ex-

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T, Ljungqvist L, Holmgren E, Paleus S, Josephson S, Philipson L, Uhlen M. Production of specific antibodies against protein A fusion proteins. EMBO J 1986; 5:2393-2398. 233. Windass JD, Newton CR, De Maeyer-Guignard J, Moore VE, Markham AF, Edge MD.The constructionof a syntheticEscherichia colitrp promoter and its use in the expression of a synthetic interferon gene. Nucleic Acids Res 1982; 215639. 234. Tabor S, Richardson CC. A bacteriophage Ti' RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci USA 1985;82:1074-1078. 235. Brown WC, Campbell JL, A new cloning vector and expression strategy for genes encoding proteins toxic to Excherichia coli. Gene 1993; 127:99103. 236. Wang RF, Kushner SR. Constructionof versatile low-copy-number vectors for cloning, sequencingand gene expressionin Escherichia coli. Gene 1991; 100:195-199. 237. Mandecki W, Hayden MA, Shallcross MA, Stotland E. A totally synthetic

plasmid for general cloning, gene expression and mutagenesis inficherichia coli. Gene 1990; 94:103-107. 238. Maniatis T, Ptashne M, Blackman K,.Kleid D, Flashman S, Jeffrey A, Maurer R. Recognition sequence of repressor and polymerase in the operators of bacteriophage lambda. Cell 1975;5:109-113. 239. Gilbert W. Starting and stopping sequences for the RNA polymerase. In: Losick R, Chamberlin M, eds. RNA polymerase. Cold Spring Harbor, N Y : Cold Spring Harbor Laboratory 1976:193-205.

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240. Pribnow D. Nucleotide sequence ofan RNA polymerase binding siteat an

early T7 promoter. Proc Natl Acad Sci USA 1975; 72:1069-1072. 241. Schaller H, Gray C, Herrmann K. Nucleotide sequence of an FNA polymerase binding site from the DNA of bacteriophage fd.Proc Natl Acad Sci USA 1975; 72~737-741. 242. Dickson RC, Abelson J, Barnes W M , Reznikoff WS. Genetic regulation: The Lac control region. Science 1975; 187:27-35. 243. Bennett GN, Schweingruber ME, Brown KD, Squires C, Yanofsky C. Nucleotide sequenceof the promoter-operatorregion of the tryptophan operon of Escherichia coli. J Mol Biol 1978; 121:113-137. 244. Maniatis T, Ptashne M, Barrel1 BG, Donelson J. Sequence of a repressorbinding site in the DNA of bacteriophage X. Nature 1974; 250:394-397. 245. Walz A, PirrottaV. Sequence ofthe P, promoter of phageX. Nature 1975; 254:118-121. 2 4 6 . Chang C N , Kuang W-J, Chen EY.Nucleotide sequence ofthe alkaline phosphatase gene of Escherichia coli. Gene 1986; 44:121-125. 247. Overduin P, Boos W, Tommassen J. Nucleotide sequence ofthe ugp gene of E. coli K-12: Homology to the maltose system. Mol Microbiol 1988; 2: 767-775. 248. Schell MA. Homology between nucleotide sequences of promoter regions of nah and sal operons of NAH7 plasmid of Pseudomonasputida. J Bacteriol 1986; 166:9-14.

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2 Gene Expression in Recombinant Bacillus Matti Sarvas National Public Health Institute, Helsinki, Finland

1 INTRODUCTION

Bacilli have many features attractive for a microorganism to be used as a host for the production of heterologous proteins; indeed, bacilli may be an optimal hostfor a numberof applications inboth industry and research. Intensive development on the expression of heterologous proteins during the last10 years has now made this mode of production feasible, and the first stepsto introduce the methods to established industrial technology have been taken. Bacilli have long been used for industrial production, the main products being secreted enzymes suchas amylases and proteases. This has provided much basic knowledge and practical technology for the culture, fermentation, and downstream processingof products. An important feature of all bacilli used in practical applications is their apathogenicity and the well-proved safetyof appropriate industrial processes using them. Most bacilli used in industry and research lack the cellular components or metabolic products toxic to humans or animals, an important feature facilitating the production of proteins of medical interest and an additional 53

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element of safety withthe technology of genetic engineering. In particular, as gram-positive organisms, bacillido not contain endotoxins (lipopolysaccharide), which are ubiquitous in all gram-negative bacteria, including Escherichia coli, and difficult to remove from many proteins in the process of purification. The prototypespecies of bacilli,Bacillus subtilis, has been characterized extensively both physiologically and genetically, second only to E. coli. This wealth of information provides a sound basisfor the genetic engineering of the bacterium for industrial applications, to develop novel modesof production, and to extend the exploitation of Bacillus as a model organism for the advanced understandingof bacterial cell biology. The long availabilityof effective genetic exchange methods, including physiological transformation, has providedan ideal systemfor the study of a broad range of genetic, biological, and physiological properties of Bacillus, resulting in a wealth of knowledge concerning the genetic map, the expression and regulation of bacillar genes,and the regulatory mechanisms associated withthe adaptation to environmental changes, including sporulation (for a comprehensive treatise, see Ref. 1). There is at present a multinational projectto sequence the entire genome ofB. subtilis (2). The development of host-vector systems has been expansive, and thereare now available a multitude of different types of plasmid for cloning and for constructing expression systems(3). The secretion of proteins, however, the is feature of bacilli that has attracted mostattention in biotechnologyand dominated their role as hosts for the productionof heterologous proteins. Most Bacillus spp. secretea multitude of proteins into the growth medium; many strains or species used in industrial processes secrete large amounts of many degradative exoenzymes, such as proteases, amylases, and nucleases. The prospect of exploiting this capacity in order to produce heterologous proteins by secretion has been the impetus for much of the research and development on Bacillus. The secretory production couldoffer attractive advantages for industrial production. Purification of a secreted protein is simpler and more economicalthan thatof a product produced intracellularly, the prevalent mode of production in most microbial production systems. A secreted protein, at least one without the embellishments may be found in eukaryotic proteins, couldbe expectedto adopt its nativeconformation even at a high levelof secretion, while effective intracellular expression often leads to the aggregation of the protein in a denaturedform; in such cases recovering the protein in an active or pharmacologically acceptable form may be tedious, if feasible at all. Secretory proteins can alsobe tailored

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to have the native N-terminalstructure more readilythan those produced intracellularly, as describedlater. Some proteins with biological activity harmful to the cell, if presentin the cytoplasm, may be produced only in a secreted form. this goal achieThe development of bacillar genetic engineering has made vable; most of the production systems developed for heterologous proteins in Bacillus are, indeed, based on secretory expression. Several production systems have been described that produce heterologous proteins Bacillus as a production host has also at high efficiency. However, using met major obstacles, the most prominent ones being the proteolytic degradation of many proteinsand the poor compatibilityof many nonbacillar proteinsfor secretion inBacillus. Much of the work on bacillar expression has dealt with development of the technology to tackle these problems. On the other hand, the secretory expression systems have'been developed exploitingthe expanding knowledge of the molecular biology of of Bacillus. The concomitant advances in gene technology have paved the way for the development of nonsecretory expression systems and have contributed much to the understandingof heterologous gene expression in other situations. in this chapThere is, therefore, much emphasison the secretion systems ter, which summarizes the technology of the expression of heterologous proteins inBacillus. Most of the discussion is focused onB. subtilis, the organism used in the vast majority ofthe studies described inthe literature, with somenotable exceptions. The well-developed methods of genetic engineeringare, in general, applicable onlyto B. subtilis, although it at present. In is not aBacillus species widely usedin industrial applications this chapter we describethe major production systems, their potential applications, and the constraints. Heterologous gene expression, the secretion of proteins inBacillus, and the genetic and molecular biological methods used in Bacillus have been the subject of several recent reviews (4-8).

2 SECRETION OF HETEROLOGOUSPROTEINS IN BACILLUS 2.1

Molecular Basis of the Secretion of Heterologous Proteins in B.subtilis

The overall pattern of protein secretion in Bacillus, studied almost exclusively in B. subtilis, follows the principle established initially for the secretion of eukaryotic proteins across the endoplasmic reticulum, and

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subsequently for E. coli, the most completely characterized bacterid model system. (For recent reviews, see Refs. 6 and 9,lO-12.) The proteins secretedare synthesized as precursors withan amino terminal extension, which contains the signal sequenceor the signal peptide. They are translocated acrossthe cytoplasmic membrane ofthe bacterial cell vectorially withthe concomitant removal of the signal sequence. Many, if not all, of the cellular components involved in this mechanism have already been identified E. in coli, with some understanding of their mode of function (for a review, see e.g. Ref. 9). The earliest event in secretion in E. coli is the interaction of the nascent polypeptideof the secreted protein with the cytoplasmic proteins belonging to the classof molecular chaperones. These includethe SecB protein (13-16) and heat-shock proteins, suchas GroEL (17-19) and DnaK (20), as well as the bacterialanalog of the signal recognition particle (11,21-23). These proteins affect the bondingof the precursorto keep itsconformation loose and compatible with the later stages of the process (9,24-26). One function of the signal peptide is to interact with the rest of the precursor molecule together withthe chaperons to achieve and maintain aconformation compatible with secretion. The peripheral membrane protein SecA (27-29), a protein with ATPase activity, specifically binds the signal sequence. This is the first step in guiding the polypeptide chainto the complex of integral membraneproteins that is known to contain a closely associated pairof SecY and SecE proteins and in addition at least the integral membrane proteins SecF and SecD (9,30-33). In a way not yet elucidated, the interaction of the signal sequence withthis complex'resultsin the translocation of the precursor polypeptide chain across the cytoplasmic membrane, possibly through a porelike structure containing the SecY/SecE protein complex. The signal sequence is cleaved off from the precursor on the outer surface of the membrane by a specific protease,the signal peptidase( h p ) (34), followed by the release of the secreted, mature protein from the membrane. Knowledge of the components of bacillar protein secretion is much more limited and has been obtainedand has been obtained rather recently (for a recent review, see Ref. 6). Protein homolog of SecA, SecY,and SecE of E. coli have been identified inB subtilis (35-40). Bacillar secA, secY,and secE genes complement the respective mutations of E. coli to a certain extent, implyingthat their functionsare similar. Thereis sequence homology betweenthe E. coli and B. subtilis protein counterparts; SecA ofB. subtilis has been shownto contain ATPase activity(36), and the predicted folding patterns of both SecY proteins are very similar. Bacillar SecA is

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somewhat shorter and SecE muchshorter than the homologous proteins of E. coli. B. subtilis homolog ofFfh protein.and the 4.5s RNA, compo(41,42). A gene, nents of the signal recognition particle, have been identified designated sips, encoding a protein with signal peptidase activity, with weak but significant sequence homology withLep theof E. coli, has been identified inB. subtilis (43). However, the deletion ofsips did not abolish secretion completely,and it was not indispensablefor survival, unlike its homolog inE. coli. A unique componentof the secretion machinery, the PrsA protein,with no known homolog E. in coli, has recently been characterized inB. subtilis (44,45). PrsA is a lipoprotein anchoredon the outer surface of the cytoplasmic membranebut predicted riot to be embedded in the lipid bilayer of the membrane. Mutation studies suggest that it functions in alate stage of protein secretion, possibly involved the in folding of the protein in a chaperone-like fashion after its translocation and in the release of the protein from the membrane (42). The overall cellular machinery of protein secretion B. in subtilis thus resembles that of E. coli, with presumed similar functional features. However, the bacillar components identifiedso far also show distinctstructural features; the bacillar system may thus have evolved and adapted in away that could be reflected in functional properties not yet elucidated. I With regardto the feasibility of the secretion of heterologous proteins in Bacillus, the role of the signal sequence is crucial.The presence of a functional signal sequence an is absolute requirementfor secretion to take place. It contributes to the folding of the nascent polypeptidechain, it is the targetof the binding and recognition by the translocator machinery, and it interacts withthe components of the translocator in the initiation of the translocationof the polypeptide across the membrane(9). On the contrary, the mature part of the exported protein, the part released from the cell after thecleavage of the signal sequence, has no known active function in the process (for a review, see Ref.46). Studies on exported bacterial proteins have virtually excludedthe possibility of any specific sequence serving asan export signal inthe mature part of the protein (see Ref. 46 and references therein). Biochemical and genetic studies involving the in vitro translocation of secreted proteinsinto vesicles of cytoplasmic membranes have not provided evidence of specific interactionof any part of the mature protein withthe components of the export machinery. The foregoing considerations suggest a model for the basic strategy in It was followed in allefforts to secrete heterologous proteins Bacillus. anticipitated that the fusionof a signal sequencefunctional in B. subtilis to the DNA sequence encoding the protein of interest would result in its

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secretion from the cell, provided that there were also functional expression signalsfor transcription and translation. Indeed,was it demonstrated very early that insulin was secreted across the cytoplasmic membrane of E. coli when fused to the signal sequenceof an exported protein, the periplasmic 0-lactamase (47). It was hopedthat a potentially large number of proteinsbemight secreted this way in Bacillus. Well-defined constraintswere also recognized. Many hydrophobic stretchesof significant length are expected to function as socalled “stop transfer”sequences (10) and arrest secretion; such sequences are likely to be found in membrane proteins,but they may also be found in some soluble cytosolic proteins. Other types of limitationsare exemplified by extensive studies on the cytoplasmic protein 0-galactosidase of E. coli. When this proteinis expressed as a fusion with a signal peptide of a secreted protein, it is arrested in the cytoplasmic membrane ofE. coli (48). At a high level of expression,the effect is also harmful to the cell. There is no defined segment(s) of 0-galactosidase, which hinders or slows down its traversion, but rather such structural features are distributed all along the moleucle (49). An interesting hypothesis has been presented by MacIntyre and Henning (46). They postulate that a crucial feature of cytosolic proteins is their rapid folding to tight conformationto prevent their unintentional export. a signal peptide were not Indeed, most cytosolic proteins provided with exported fromE. coli efficiently (50). A more subtle restriction on secretion may be posed by the presence and location of charged amino acid residues in the mature part of the protein. Basic amino acid residues at the amino terminusof the protein elicit especiallydramatic hindrance to secretion in bacteria(51-54). In agreement with this observation, secreted bacterial proteins tendto lack lysyland arginyl residuesat the amino terminus ofthe native protein, and acidic and basic residues often occur in a paired manner (935). Proline is never found at the N-terminusof secreted proteins, and when introduced, completely preventsthe cleavage of the signal peptide in E. coli (56,57). These restrictions clearly indicate that proteins that are secretory by nature are the primary targetgroup of heterologous proteinsto be produced withthe secretory mode inBacillus. Fortuitously, a large number of proteins of either industrial or pharmaceutical interest are secretory proteins. The foregoing principles have been applied in a number laboratories of to develop systems to secrete heterologous proteins inBacillus. In most studies the host organism has beenB subtilis, and combinations of variAs ous expression signals, signal sequences, and plasmids have been used.

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discussed in more detail in the following pages, these studies have demonstrated that the principle is applicableto the production of a number of proteins, both model proteins and those of practical interest,but the range of the types of proteins secreted efficientlyBacillus in is unexpectedly narrow.

2.2 Secretion Systems Based on the Genes of Major Exoproteins of Bacillus spp.

2.2.l

Vectors Based on or-Amylases

The most straightforward approach to achieving the secretion of a heterologous protein has been to insert the structural gene of the protein of interest downstream from the expression signalsand the signal sequence of a bacillar gene encoding an exported protein. The genes of exported amylases and proteases are an obvious choicefor this purpose and have been utilized in most approaches. Both types of enzymes are secreted at high levels in many species ofBacillus (58,60), implicating the presence of effective promoters, and signal sequences of high compatibility with the secretion machinery.Furthermore, due to industrial interest,mutant strains with increased production and enhanced level of expression of these genes have been developed and are available. The first practical system, also thoroughly explored, was the one based on the a-amylase of B. amyloliquefaciens. Many strains of this species naturally secrete high levels ofa-amylases and proteases (60). To develop a secretion system, Palva et al. (61) initially clonedthe amyE gene of an industrial strain encoding a-amylaseto the plasmid PUB1 10;the multicopy cloning vectorof staphylococcal origin used extensively as a vector for cloning and expression B. insubtilis (3).Up to 1 g/L of a-amylasewas secreted into the growth medium when this hybrid plasmid was expressed in B. subtilis (61). This is 2500-fold more than the endogeneous level of a-amylase secretion inB. subtilis, showing both the effectiveness and compatibility of the expression signals ofB. amyloliquefaciens in B. subtilis and the capacity of B. subtilis for highly increased levels of secretion. Furthermore, the hydrid plasmid was very stable. Insertion of DNA fragments of various origins into derivatives of pUBllO often results in unstable constructions (62), presumably due to the rolling-circle replica(3). However, inthe case of thisamyE intion of this family of plasmids sert, there was hardly any structural or segregational instability of the construct (63), even when grown over 50 generations in the absence of selection pressure. Similar stability was a characteristicof many derivatives of this hybrid plasmid constructed later (below).

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The feasibilityof basing the secretion of heterologous proteins on the expression and secretion signals of thisamy gene was demonstrated by fusing the segment of DNA encodingthe mature part of the periplasmic TEM P-lactamaseof E. coli downstream from the signal sequence in the hybrid plasmid above. A set of plasmids was constructed withthe fusion Of the P-lactamase gene with a linker sequence a few of codons (providing a cloning site) either directlyto the end of the signal sequence or a few codons downstream (64). The P-lactamase was well expressed from all constructions. Meeting expectations, virtually all of the enzyme was secreted into the growth medium; lessthan 5% of the P-lactamasewas cell bound in the form of the expected size of the precursor. The signal sequence was cleaved off from the secreted form; the cleavage took place in all constructionsat the end of the signal peptide,at the "natural" cleavage site. Levels of more than 20 mg of secreted P-lactamase in 1ofLthe culture supernatant were obtained inthe early stationary phase of growth in shake flask cultures ina rich laboratory medium; a good level of expression, although much lower than that of the a-amylase itself(see later for a discussion of possible reasons). P-lactamase was secreted inthe exponential phaseof growth and continued into early stationaryphase, fol-. lowed by a decrease inthe amount of the enzyme inthe medium (64,65) (for proteolytic degradation, see below and Section 2.6). This pioneering study encouragingly demonstratedthat a heterologous protein can be secreted efficiently B. in subtilis by usingthe elementary strategy of fusing to it a bacillar signal sequence and expressing it from a highly active promoter. Derivatives ofthe plasmid above have been developed to provide a set of secretion vectors withan appropriate cloning site (HindIII in most members of the set)and with properties enabling the ready expression of a gene or a DNA fragment encoding the protein of interest. The expression signals ofthe secretion vectorare in an insert of about 500 bp in the BamHI site of PUB1 10;the insert contains the promoter, the ribosome binding site, and the signal sequence of the amy gene of B. amyloliquefaciens. Upstream from the promoter, the fragment alsocontains an inverted rewhich prevents peat region functioningas a transcription termination site, any possible read through and interference with the expression upstream. Immediately at the end of the signal sequence there is a synthetic oligonucleotide with the cloning site, HindIII, with zero, one, or two extrabases between the linker and the signal sequence in different members of the set. Thus, by an appropriate choice, the gene insertedat the HindIII site will betranslated in the same reading frame with the signal sequence. The authentic cleavage site ofthe signal peptide, after its last Ala residue,is

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preserved. The linker oligonucleotide encodes three or four amino acid residues, depending on the particular construction or reading frame. In the basic setof plasmids, the HindIII linker is followed by PUB110 DNA, thus the translation and transcription termination signal should be provided by the insertat the HindIII expression site.In a second setthere is, downstream from the HindIII linker, another synthetic oligonucleotide with a translational stop codon in all reading frames(66). In a third set there is a further extension, a fragment with the transcription termination site of the amy gene (67). Efficient termination of transcription may, although not invariably, increase the efficiency of expression and even the stability of the plasmid. m e applicabilityof this secretion vector system, with some minor variation in the structure and the length of the linkage sequence betweenthe signal sequenceand the protein to be produced, has since been tested in several studies.A pattern has emerged which, by and large, has also been found withother secretion vector systemsto be described below. Interestingly, to date this secretion vector system has hardly been surpassed in terms of effectiveness and in the range of applicability. Penplasmic proteins of gram-negative bacteria, some of them of inductrial interest, such as pectin methylesterase ofErvinia chrysanthemi(68), or those of pharmaceutical interest, like somesubunits of the pertussis toxin (59,69), have been secretedrather efficiently, up to 500 mg/L of culture medium under of the signal optimal conditions. Inall of these constructions the cleavage peptide left someamino acids derivedfrom the linker on the N-terminus of the secreted protein.Partial proteolytic leavageof the productwas also found in some cases (69). Some periplasmic proteins were, however, either not expressedor not secreted,as demonstrated by very poor levels ofproduction of some of the subunits of the pertussis toxin (59). Not surprisingly, considering their closer relationshipto B. subtifis,and thus their expected better fit to the secretion machinery, extracellular or secreted proteins of gram-positive bacteria have been both expressed and secreted at high levels. For example,the thermostable a-amylaseof B. licheniformis is producedat levels morethan 500 times higherthan the amount secreted by the native host(70). However, even some secretory proteins of gram-positive bacteria, such as the inactive variant of diphtheria toxin, was produced and secreted onlyat levels of lessthan 5 mg/L, for reasons that were not obvious (71). The first eukaryotic protein expressed the with aid of this secretion system was human interferon-a2. It was secreted into the growth medium, processed by the cleaving of the signal peptide, and found to be biologi-

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cally active. Very disappointingly, however, and anticipating later findings with other secretion systems,the efficiency of its secretionwas verypoor, only in the range of 1 mg/L, withthe product being unstabledue to proteolytic cleavage (see Section 2.6 for more detailed discussion) (72,73). Another a-amylase-base secretion system has been constructed, based on the gene of the saccharifying amylaseof B. subtilis, by way of close analogy withthe foregoing setof vectors. The umyE gene from a hyperproducer strain of B. subtilis was cloned and a DNA fragment encoding the signal peptideand a long upstream segment containing the promoter and the so-calledumyR2 element, upstream from the promoter, was cloned in a derivative ofthe multicopy plasmidPUB110 (74). amyR2 was identiof fied in the hyperproducing strain as an element enhancing the activity the amyE gene (75); it has a palindromic sequenceand one could anticipate that it is a palindromic transcription termination site of the upstream gene and thus prevents transcriptional read-through by possibly interfering with the expression of umyE. Using the TEM P-lactamaseas a model protein, its processing and secretion with virtually no intracellular accumulation, were demonstrated, when the gene withoutthe signal sequence was fused downstream from the signal sequence of umyE of the vector (74,76,77). In some ofthe constructions, in additionto the signal sequence, there were about 10 N-terminal codons of the mature a-amylase, a "prosequence" foundin this a-amylase and nibbled offtheingrowth medium after cleavage ofthe signal sequence. This addition decreased the amount of secreted P-lactamase (to about half); the later finding showed that this prosequence hasno role inthe process of the secretion of a-amylase (78). Many "random" peptides of about 30 to 40 residues were also inserted between the signal sequenceand another model protein, thermostable bacillar a-amylase (79). Interestingly, some of them hardly affected secretion, suggesting much leeway the in designs of constructions for the secretion of a particular protein. The overall efficiency of this secretion vector was, however, much lower that that of the one based on the a-amylaseof the B. amyloliquefaciens. The maximal level of 0-lactamasewas only about 10% of the latter one in ordinary richlaboratory media (74). This probablyis a reflection of the lower rate of a-amylase secretion and thus presumably amyE gene activity in B. subtilis, even in the hyperproducing amyR2 strian. However, much improvementof the yield, up to 50 to 100 mg/L, was achieved in glucose and succinate rich media combined with growth under conditions of poor aeration usingan exoproteasedeficienthost (80). The maximum level of 150 mg/L of the same P-lactamase has been obtained with B.the

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amyldiquefaciens amylase-based vector in rich media with 6% of glucose (81). Efforts to produce two eukaryotic proteins, mouse interferon-/3 and a plant protein, thaumatin, with this system have been described (82-84). Again, the proteins were secretedand processed without cellularretardation, but at a very low level indeed (see also Section 2.4). Several constructions of mouse interferon were tested, with different NH2-terminal extensions. They were all secretedat similar levels, but there were severalfold differences betweenthe specific biological activities of the different constructions. In both secrtion systems baseon a-amylases, the maximal amount of the product is reachedat the end of the logarithmic growth phase in rich laboratory media such as L-broth. There is a decrease in the amount of the product in theculture medium inthe stationary phase, as indicated in detailed studies with the model protein &lactamase (80,85,86a). Unlike the case of P-lactamase, the production of homologous a-amylase, expressed from the same vector, continued for 10to 30 h after the cessation of growth (86). This suggests proteolytic degradation of the heterologous product by exoproteases abundant in theculture medium at stationary phase. The yield could be improved by usingmutant host strains with greatly (over 95%) decreased amounts of exoproteases (64,85) (see Section 2.6 for more discussion of the role of proteases). Furthermore, culture conditions suppressing the induction of exoproteases, suchas with high concentrations of glucose in the medium, can improve the yield further (81). An interesting feature of the secretion vector based on theamy gene ofB. amykhefaciens is the lack of catabolic repression by glucose when the promoter is fusedto a foreign gene (71,81).

2.2.2 Vectors Based on Proteases

Proteases are another class of bacillar exoenzymes secreted at high levels in manyBacillus species (60),thus providing a potential source of expression and secretion signalsfor the constructionof secretion vector systems. Like the a-amylase gene of a B. amyloliquefaciens,major exoprotease genes of this species have also been found to be efficiently expressed, with significant secretionlevels of the appropriate protein in B. subtilis (87,88). Several secretion vector systems have been constructed based on these genes. The activity ofthe genes ofthe major bacillar exoproteases is coupled to the initiation of sporulation. The synthesis and secretion of proteases does not start before the earlystationary phase of culture. This is a po-

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tential drawback for a secretion vector system based on these genes. The secretion of the heterologous protein follows this same pattern of temporal expression, resulting in the simulataneous secretion of the protein of interest and the exoproteases, with possible concomitant degradation. Therefore, the use of protease-deficient host strains for the production of most proteins (see Sections 2.6, 2.9) is highly desirable. On the other hand, the activity of protease genes is also regulated by several transcriptional regulatory factors, the degQ and degU among them (for a recent review, see Ref. 89). Certain mutations in these genes or their overexpression (degQ) (e.g., by cloning in a multicopy plasmid) stimulate the activity of exoprotease genes; the target sequences of these regulators are located in the region about lo00 bp upstream from the promoter. These regulatory genes provide a means to regulate, as well as to enhance considerably, the production of a heterologous protein, when secretion vectors based on protease genes are used. (deg genes are involved also in regulation of the genes of other exoproteins such as levansucrase, used as the basis of other secretion vectors; see below.) Unlike other bacillar exoproteins, the secretion of proteases in bacilli involves other extensions in addition to the signal peptide in the precursor, which are cleaved off in the process of secretion. The N-terminal segment of the precursor (often called alsopresequence), in the range of 30 amino acid residues, is a typical signal peptide with the appropriate function (for a review, see Ref. 6). Between the signal peptide and the N-terminus of the mature exoprotease, there is another extension, the prosequence, which varies in length from about 60 to 200 amino acid residues in the precursors of different proteases (6). The prosequence is essential for the maturation of the precursor to an active protease. It has recently been shown to function in the final step of folding of the protease, after its translocation across the cytoplasmic membrane but before release from the membrane, to promote its final, enzymatically active conformation (90,91). The prosequence is then cleaved off, mainly autocatalytically, releasing the active enzyme from the cell. Consequently, mutant exoproteases, which are enzymatically inactive, are not released from the prosequence and secreted. This correlates with greatly decreased synthesis (92,93). Although it was demonstrated subsequently that the prosequence does not have a function in the process of secretion of BaciZZus proteases (94), such a role was considered likely when many early and successful secretion vectors based on proteases were designed, resulting in vectors containing both a signal sequence and elements of the prosequence. However, as described below, segments of prosequence are dispensable for the construction of secretion vectors.

Gene Expression in Recombinant Bacillus

65

The first approach was taken with the neutral protease gene (npr)of B. amyloliquefaciens. In the initial construction a fragment of DNA encoding the signal sequence plus the entire prosequence (“preprosequence”), and the promoter region with upstream region of a few hundred base pairs was cloned in the plasmid PUB1 10 (88,97). The gene encoding the a-amylase of B. subtilis (without its signal sequence) was fused downstream from the prosequence, with the first 15 codons of the mature part of the protease intervening. Good expression of the a-amylase construction was seen in B. subtilis. Furthermore, the long prosequence (209 amino acid residues) was not present in the secreted a-amylase. Interestingly, the prosequence did not seem to interfere with the folding of a-amylase, from which was removed effectively despite the obvious lack of an autocatalytic process, presumably by exoproteases. An analogous construction based on the promoter region and preprosequence of the alkaline protease of B. amyloliquefacienshas also been described (95). Secretion of the protein A of Staphylococcusaureus was demonstrated with this system (96). However, in this case, the prosequence remained fused to the secreted product, although cleavage of the signal sequence took place. Reasons for the difference between these findings have not been elucidated. The secretion of two human secreted proteins, interferon-@(IFN-@) and growth hormone (hGN), has also been studied with the foregoing npr-based vector system. The parts of the appropriate genes encoding the mature protein were fused to the 3’ end of the prosequence as in the model proteins above (97,98). In good agreement with the studies of model proteins, both human proteins were secreted, and again there was no prosequence left in the secreted protein. However, the amounts were much smaller than those of either a-amylase or another model protein, P-lactamase. The amount of hGH was estimated to be about 1.5 mg/L of culture. Furthemore, proteolytic degradation of both proteins was evident. This secretion vector was developed further by the removal of most of the prosequence, resulting ultimately in a construct with the promoter and its upstream region, but with secretion signals composed of the signal sequence plus only the first 21 codons of the prosequence, followed by the structural gene encoding the protein to be secreted. In agreement with the lack of role for the prosequence in secretion per se, this vector performed similarly to the one with complete prosequence, with respect to the secretion of both a-amylase and hGH, in terms of the amount of these proteins secreted as well as the removal of the short N-terminal fragment of the prosequence (99,100). It was further demonstrated that the npr promoter of the vector was susceptible to the control of DegQ. Introducing

66

Sawas

another multicopy plasmid with the degQ of B. amyliquefaciens to the production host increased the expression of a reporter gene (cat) and the secretion of hGH moderately, about 1.5-fold (101). The use and development of this secretion system for the efficient secretionand production of hGH is described in Section2.4. Severallaboratories have described secretion based both on the neutral protease geneof B. amyloliquefaciens and on the alkaline protease gene of the same species, where the only secretion signal is the signal sequence. The signals of npr have been used in the vector constructions described in Refs. 95 and 102-105, the apr in Refs. 95 and 102 and a hybrid with npr promoter and the signalsequence of the subtilisinin (106). The plasmids were multicopy plasmids, either replicating onlyin Bacillus, or shuttle plasmids replicating also in E. coli (102). Some of these plasmids, like those based on either npr or apr in Ref. 102, were tailoredto secretion vector systems with a convenient cloning site immediately downstream from the cleavage site of the signal sequence, allowing the construction of fusions whose processing during export could be expected to release a secreted protein withthe native N-terminus of the heterologous protein. Studies of model proteins of bacterial origin, protein A of S. hureus (95) and the extracellular form of the a-amylaseof B. lichemifonnis (103), showed that the signal peptide alone was a good secretion signal resulting in efficient secretion,up to 3 g/L of protein A, and 140 mgof a-amylase under optimal culture conditions. Whether the signal peptidewas cleaved exactly as intened, at the native cleavage site,was not possible to determine. The secreted proteins had ragged N-termini, most likely to dueproteolytic cleavage inthe culture medium, which was quite evident in most cases. As far as bacterial proteins are concerned, the efficiency of secretion by vectors based on the protease genes of B. arnyloliquefacens is, by and large, similarto that observed with vectors based on a-amYlase described above, although there are data only on proteins from grampositive bacteria. The secretion and production of a number of eukaryotic and human ~ U ~ U ' I proteins have been explored in these secretion systems, including atrial natriuretic factor (hANF) (107), human growth hormone @GH) ( l a ) , bovinepancreaticribonuclease (76), prochymosin and human IL-IP (105) (for details, see also Table 1).The secretion of these Proteins was achieved,but as described in Section 2.4, the efficiencyWaS generally poor. The feasibility of the secretion of a eukaryotic protein with a native N-terminus and biological activity was demonstrated both for the Pan-

Gene Expresston In Recombinant Bac///us

67

creatic ribonuclease and for hGH, with yields of about 5 and 8 mg/L, respectively (thelatter increased to 40 mg/L in highdensity cultures).

2.3

Other Secretion Vector Systems

A set of secretion vectors and production system has been constructed

exploiting the expression and secretion signals of the levansucrase gene Ivs of B. subtilis (108-113).The primary emphasis ofthis set has beento introduce the elements of inducibilityand the activity inthe exponential phase of growth to the expression system. This has beenthe main goal in using the heterologous expression and secretion signals of the staphylokinase of S. aureus, for both secretion and intracellular expressionsystems (1 14,115). These production systems, and their use and benefits for secretory expression,are discussed in Section 3. Some secretion vectors have been based the on signal sequences ofthe a-amylase of the B. amyloliquefaciem or the apr exoprotease of B. subtilis, but fusedto promoters of other genes. A few constructions have been described, each, however, usedto express onlya single protein. The signal sequence ofamy has been combined with a promoter of the phages SPOl (1 16)and SP02 (1 17), and the signal ofapr with a strong vegetative promoter of B. subtilis cloned for the purpose (1 18). An analogous construction was made by fusinganother strong, vegetative promoter ofB. subtilis, P43, to the signal sequenceof the levansucrase ( h ) gene (1 19). The performance of these vectors, especially in comparison with the vectors based on the appropriate native genes, is difficult to evaluate, becauseno common reporter proteins have been studied. Three constructions were tested with an eukaryotic protein. The peak productionswere the secretion of 5 mg of a single-chain antibody molecule and 10 mg of a plant galactosidase per liter of culture, with combinationsof the P43 promoterIvs signal (1 19) and the SPOZamylase signal (1 17), respectively. The E. coli P-lactamase was reported to have been secreted very efficiently with the vector combiningthe secretion signalof apr with the vegetative promoter, but data on absolute amounts were not reported (1 18). An early secretion vector construction was based on the gene of the plactamase (penicillinase) ofB. licheniformis.This proteinis an exported lipoprotein, anchoredon the outer surface of the cell membrane by three fatty acid molecules covalently boundto the N-terminus ofthe molecule (for a review, see Refs. 120 and 121). Considerable amounts of the protein domain of this moleculeare released in a soluble and enzymatically active exoform inthe growth medium inthe stationary phase of growth

68

Sawas

by a proteolytic cleavage near the N-terminus. When clonedB.in subtilis, the protein is well expressed, but there is proportionally much less ofthe exoform as in B. licheniformis(122). To construct a secretion vectorthe promoter and signal sequence of the penP gene were used; the performance of the vector was studied by fusing these expression signals to DNA sequences encoding preproinsulin, human @-interferon, and growth hormone (123). There was a very low level of secretion of insulin together with cell-bound protein, cell-bound interferon, and an undefined level of secretion of growth hormone.The performance of this vector system has not been tested with reporter a protein secreted efficiently with some other secretion vector system.

2.4 Secretion of EukaryoticProteinsin BacNus

As described inthe sections above, heterologous proteins of bacterial origin are, as a rule, secreted inB. subtilis with the aid of the secretion vectors. Those of gram-positive speciesare secreted very efficiently, as are many of thosefrom gram-negative bacteria. Incontrast, eukaryotic proteins, particularly those of human origin, even when they are secretory proteins by nature, are invariably secreted at much lower levels. Many eukaryotic proteins areof pharmaceutical or industrial interest, and much work on the bacillar secretion vectors hastherefore been on eukaryotic proteins. None of bacillar secretion vector-host systems have found been to be clearly superiorto the others for the productionof eukaryotic proteins, but inthe course of these studies, elementsfor optimizing the secretoary modeof production have been identified, resulting in improvements in the efficiency of secretion and yields suitablefor the industrial production for some proteins. In the following pages, some findings relevant to the efficiency of secretion of eukaryotic proteins in Bacillus will be discussed, the constraints identified, and the means for optimizing the vector-host systems and the conditions of production presented. The potential of secretion vector systemsto produce eucaryotic proteins has been tested with interferon (IFN) in many laboratories. Interferons are naturally secreted proteins and of relatively small size(about 20 kDa); botha- and @-interferons are nonglycosylated. These are all features expectedto be favorablefor secretion in a bacterial system. Furthermore, there would be much practical interest to produce these proteins in a bacterial production system other than E. coli (for references, see Ref. 72). To study the secretion vector based on the a-amylase B. amyof loliquefaciem, Palva etal. (72) and Schein et al.(73) inserted the segment

Expression Gene

in Recombinant Bacillus

69

of DNA encodingmature secreted humaninterferon-a2 (IFN-a2) inthe vector. In the first construction, there were a few linker-derived codons between the cleavage site of the signal peptide and the N-terminus ofIFNa2; in the latter construction, the IFN-a2 insert was fused directlyto the last codonof the signal sequence. Both constructions were expressed, and secretion ofIFN into the growth medium was clearly demonstrated. More than 90% of I F N synthesized wasfound in the culture medium.Furthermore, in both cases there was accurate cleavage of the signal peptide at its C-terminus. Thus B. subtilis is clearly capable of secreting this eucaryotic protein. However, the amount of IFN inthe growth mediumwas only in the range of1 mg/L of culture, while expression levelsup to 1 or 0.1 g/L had been obtained in similar growth conditions.(L-broth, shake flask culptures) inthe case of homologous a-amylase or heterologous, bacterial lactamase (TEM of E. coli)respectively (61,64,65,73). The strainused for the experiment wasa mutant with much reduced secretion of exoproteases; in agreement with protease sensitivity of the interferon, hardly any activity found in culture media when the host strain was wild type. Quantitatively similar results were obtained in experiments designed to secrete humanand mouse IFN-fl inB. subtilis using secretion vector systems basedon the neutral protease geneof B. amyloliquefaciens [human IFN-p (97)] or the a-amylase gene of B. subtilis [mouse IFN-b (82,83)]. Again, active interferonwas secreted, but far below the secretion capacity of the system,as judged by the level of secretion of homologous or heterologous bacterial exoenzymes expressed from the same signals (74,97). The amount of IFN secreted in the production system of Palvaal.et(72) was not higher than that in the others, despite its much higher inherent expression capacity. No secretion, onlya low level of intracellular accumulation of murine INF-a7, was achieved with a secretion vector based on the levan sucrase promoter and signal sequence(108). Thus rather similar levels of secretion were found for these eukaryotic proteins, although the efficiency of the promoters used varied considerably and the signal peptides were of differentamino acid sequences. Adjustingthe fusion of the murine IFNto the signal peptide in such a way that its predicted native secondary structurewas maintained also did not improve secretion. Clearly, there are severe obstacles, either for the secretionor expression of IFN itself in these systems. A somewhat different pattern was observed by Breitling et al. (114) with a secretion vector based on staphylokinase (above). Human IFN-a1 was secreted up to a level of 15 mg/L of culture in ordinary L-broth. This is about 10-fold higherthan obtained with the a-amylase secretion system;

70

Sarvas

IFN-a1 is closely related to IFN-a2 in its sequence and properties. Furthemore, the level of IFN secretion was only slightly below the level of secretion of staphylokinase itself using these same expression signals in B. subtilis under similar growth conditions(about 25 mg/L). As pointed out above [p.67 and in (114)], expression from the sak vector takes place effectively in the exponential phase of growth mitigating the effect of exoproteases, which might contribute to the better yield observed (but see also the role of degradation below). Alternatively, the combination of the sak signal peptide with IFW is especially favorable. It will be interesting to see whether this vector systemcan be adjusted for even higher levels of secretion of IFN or other heterologous proteins,up to the level of industrial significance. To date, the secretion B. in subtilis of a large numberof other eukaryotic proteins of different origin has been studied withdifferent secretion vector sytems. Table1 shows a partial list of representative experiments. As in the case of interferons,the gene of interestwas inserted into these secretion vectors eitherto result in a fusion with an exact joint to the signal sequence,or often with a few intervening codons derived from linkers with cloning sites (usually with little or no effect on the secretion). In most of these studies, the production host was also a mutant with decreased amounts of exoproteases, and production of the secreted product was reported for shake flask cultures in richlaboratory media. The overallpattern is not much different from that seen abovefor interferons, with a few notable exceptions. Regardless of the size, origin, or natureof the protein(e.g., glycosylation ofthe native protein), the level found in the culture supernatant at peak level of secretion was low in the majority of cases, from traces (indicated bya + in Table1) to a few milligrams per liter of culture. Again, the amount was always far below the amount of a homologous proteinor a bacterialreporter protein secreted by the same system; a difference of upto 1000-fold could be estimated. As a rule, the signal sequencewas absent from the product secreted; depending on the system, the heterologous protein was released witha native N-terminus or with a few additional or missing N-terminal amino acid residues (in most studiesthere was no effort to trim the fusion for exact cleavage). Most proteins with assayable biological activity had retained that activity. Rather consistently,the presence of a cell-bound precursor with an uncleaved signal peptide was either demonstratedor deduced, but the amountwas small in relation to the potential expression capacity of the system. Althoughthe overall level of secretion of the heterologous proteins was low, there were more than 2000-fold differences in secretion levels

~ecrgtionvector s ~ s t e ~ 67 Viral ~ e ~ b r a n47~ ~lycop~otei~se Bovine 40 pro~h~osin Sin~le-ch~n 26 rgc~~binant

+ + and 1 0.1 5

, a-amylase

+ +

P43 and ss of

.rap$gene of B. U m Y i o ~ ~ ~ M e ~ ~ ~ i e ~ P and ss of amy of ~20,221 B. f f ~ y i u ~ ~ ~ u e f u ~ i ~ ~ 106 P of the npr and ss of apr of B. s ~ b t i ~ ~ P 119

-

-;I 111.

0

tib body

Human lysozyme

14

0 or 0.2

Bovine pancreatic ribonuclease H u ~ a ~ p roinsulin man atrial na~ri~retic factor Plant protein t~au~atin

13

1-5

140, @-lactamase P and ss of npr, and P of spol or P1 with ss

+

116

of umy of B. s u b t ~ ~ ~ s 76 P and ss of u p of B.

a~y~~li~uefu~~ens 9

0.1

3

0.5

22

+

-

Protein Plant galactosidase

Size (kDa) 40

Amount secreted mg/L of cultureb 10

Human IL-1/3

18

7

Human IFN-P

18

+

Human growth hormone

22

50

Biological activity secreteC(mg/L)

+

Secretion vector system based ond Refs. SP02 promoter, ss of 117 amy of B.

amyloliquefaciens

+ 1O00, neutral otease , neutral protease

P and ss of npr of B. subtilis P and ss of npr of B.

105

97

amyloliquefacien P and ss of npr of amyloliquefaciens

bCulture in rich laboratory media in shake flasks if not indicated otherwise. ‘Judged by the secretion of a homologous or bacterial model protein under similar or nearby similar conditions. a +indicates the secretion of a small amount detected either with immunoblotting or by enzymatic activity. dDesignations of the genes: m y , a-amylase; apr, alkaline protease (subtilisin); npr, neutral protease. P and ss stand for promoter and signal sequence, respectively. %xtra membraneous domains of vesicular stomatitis virus membrane glycoprotein G and Semliki Forest virus glycoprotein E l .

Expression Gene

in RecombinantBac///us

73

among different proteins. Onecannot single out any obvious parameters correlating withthe efficiency of expressionand the secretion of a particular protein. It is thus interesting, and also of practical significance, that there were also cases of remarkably efficient secretion. Good production of human growth hormone (hGH) has been achieved (99,101) with the secretion vector based on the neutral protease gene (npr) of B. amyioliquefaciens (above), seemingly by maximizing the level of transcription and optimizing growth conditions. The insert encoded the mature hGH, with a short oligonucleotide (derived from the prosequence of npr) between the end of the signal peptide and the N-terminal codon of hGH. This construction resulted initially inthe secretion of the peak level of about 10 mg/L in a protease-deficient strain of B. subtilis. Again, this rather modest level isfar below the potential of the system, representedby hundreds of milligrams of the neutral protease B. of amyioliquefaciens under similar conditions(88). However, about a five-fold increase was obtained when the degQ gene ofB. amyloliquefaciens was introduced into the production host as a chromosomal insertion. Finally, by shifting production to fermentor cultures and applying a procedureof transferring the cells to fresh mediumat late stationary phase of growth (101), up to 200 mg of hGH per liter of culture was secreted, hGH comprising about 30% of total extracellular proteins. The majority of the protein was of the size of but native hGH (22 kDa), withthe signal peptide presumably cleaved off leaving a ragged N-terminus. Despite the use of a low-exoprotease strain, there was a considerable amount of a degradation product of about 15 kDa. The same vector system was also trimmed to fuse hGH gene directly to the end of the signal sequence(104). This modification didnot affect the level and pattern of expression significantly:about 10 mg ofhGH/L was found in the late stationary phase in shake flask cultures. In this construction, need for the termination of transcription of the inserted gene was also clearly demonstrated. Without a termination sitethe yield was less than 10% of the one above. The secreted hGH had the specific activity ofthe native hormone,and most important, its N-terminal sequence matched the authentic one. The authors achieved production up to 40 mg/L of culture in a high-density culture. Human interleukin-3 (IL-3),a 13-kDa glycoprotein, isan example of another eukaryotic protein, which has been produced successfully at high levels in the culture medium of Bacillus by choosing the proper production strain and culture conditions (124). The hostwas an industrial strain of B. licheniformis, which is an effective secretor of a-amylase, devoid

74

Sawas

of the major exoproteasesand nonsporulating. The secretion systemwas a derivative of the pUBllO plasmid, with the expression cassette composed of the signal sequence ofthe a-amylase of B. licheniformis, combined either with the promoter of the same gene or with a promoter of was introduced the plasmid PUB110. The sequence encoding native IL-3 into the vector by precise fusion to the signal sequence. Upto 200 mg/L of IL-3 protein and about 100 mg/L of nondegraded IL-3 was found in in the culture medium in a fermentor batch; virtuallyno IL-3 protein remained in the cellular fraction. Secreted IL-3was fully active biologically although it remained unglycoslated. A purification procedure compatible with industrial production was developed, and the purified product has now entered clinical trials.Van Leen etal. (124) also studied and evaluated the production of IL-3 in E. coli, Saccharomyces cerevisiae, and mammalian cell production systems.In terms of yield, quality, and production cost, they judged the B. licheniformis system to be the most suitable one for practical production.

2.5 ConstraintsontheSecretion of Heterologous Proteins in Bacillus The overallpattern emerging from the studies described above is that secretion of many heterologous proteins of bacterial origin with the secretion vectors isquite efficient, but some bacterial proteins and most of the eukaryotic proteins are produced with low and variable yields. The studies performed so far give few indications as to whether this high degree of variation is relatedto the type of protein or someof its specified properis not very high and most ties. However, the number of proteins studied of the eukaryotic proteins had biological activityof pharmaceutical interest; they may comprise a biased group. Reasonsfor the inefficient production and some possible ways to improve it are discussed below.

2.6 ProteolyticDegradation of Heterologous Proteins Secreted in Bacillus B. subtilis and other Bacillus species typically secrete proteasesinto the growth medium (for a review, see Ref. 58). In B. subtilis, the alkaline, serine protease, subtilisin,and the neutral metalloprotease comprise more than 90% of all secreted exoproteases. They are efficient enzymes with low substrate specificity, especially subtilisin. In addition, there are several minor proteases (see below). The expression of proteases is temporally

Gem Expression in Recombinant Bacillus

75

controlled; the maximal synthesis takes place in the late exponential phase of growth, concomitant withthe initiation of sporulation. Most ofthe heterologous proteinsof interest, which are candidates for production inBacillus, are sensitive to these exoproteases,and generally much more sensitive than bacillar exoproteins. An obvious and anticipated reason for the low extracellular level of heterologous proteinsproduced with secretion vector systems could thus be (rapid) proteolytic degradation in the growth medium. This has actually been observed to varying degrees with all heterologous nonbacillar proteins secreted with any of the secretion systems described (see,e.g. Refs. 125 and 126). A clearcut, even decisive rolefor proteases in the decrease of the yield was shown for some of the model proteins. Ulmanen et al.(65) found that the peak level of P-lactamase in theculture supernatant was about 10 to 20 mg/L when secreted from the vector based onthe gene of the a-amylase ofB. amyloliquefaciens.This level then decreased rapidly inthe stationary phase of growth, suggesting proteolytic degradation. The rate of degradation correlated with the amount of the exoproteases secreted by the host strain (85). A similar decline has been shown for the same 0-lactamase in other secretion vector systems (127). Even at the peak level of secretion the amount was only inthe range of 10% of the amount of homologous a-amylase expressedfrom the same vector. However, pulse labeling experiments showedthat the rate of secretion of both proteins was identical, even in late stationary cultures, where the ratio of P-lactamaseto a-amylase (assayedas protein) was about 1500. Pulse-chase experiments demonstrated degradation of P-lactamase in the culture medium at a rate comparable to its decreased steady-state level. Thus, in the case of this model protein, the sole reasonfor the low yield compared to the homologous proteinwas clearly proteolytic degradation. Another documented example of significant proteolysis of a heterologous proteinthat is effectivelyand efficiently secreted per se B. in subtilis is the protein A of S. aureus. This proteinwas secreted at a very high level from another a-amylase vector, but wasit found in the culture supernatant to be largely in a fragmented form (128). At least apartial contribution of proteolytic degradationfor the low yieldOf many other heterologous proteinis indicated by the decreased amount of the product after extended growth, concomitant with the appearance of exoproteases in the culture medium, as describedfor some proteins in previous sections. There is thus little doubt that extracellular proteolytic degradationis a

76

Sanras

major problem for the production of heterologous proteins sensitiveto in many laborabacillar exoprotease. There has therefore been much effort tories to construct host strains devoidof proteases. Exoprotease-deficient mutants of B. subtilis have been isolated after mutagenic treatment andused as hosts for the productionof secreted proteins. Some mutations have been shown to be present in the known genes of (apr)and neutral protease(npr) the major exoproteases, alkaline protease (96,101). Others are not characterized genetically, yith single or multiple mutations affectingthe level of exoproteases, perhaps via the regulatory network affecting the level of expression of exoenzymes, as well as possibly sporulation and competence (85,97,114,129). Well-characterized spo mutants such as spoOA which affect the secretion of multiple proteases can also be used as low-exoprotease hosts(107). Most of these mutants have residual exoproteolytic activity,up to a few percent of that of the wild type. Efforts to lower that level further by mutagenesis often decreased viability, presumably via the regulatory system above. The cloning and identification of the structuralgenes of the exoproteases ofB. subtilis was a major step forward, providing the means for their precise deletion. Such deletions, even those encompassing several protease genes, turned out not to affect optimal growth. Nor did they impair sporulation, contrary to previous beliefs concerning their “essential” role in this process. However, the culture supernatant of multiple protease-negativemutants may contain more cell-derived proteinsthan that of the wild type, indicating cell lysis and hampering the purification of the secreted product(IPalva, . personal communication). The deletions, or inactivationby point mutations, of the two genes of the major proteases of B. subtilis, apr and npr, simultaneously (130-133) decreased the level of proteolytic activity inthe culture medium to 10 to U)070 of the wild-type level, dependingon the particular B. subtilis strain used. Since becoming available, mutants defective with respect to both of these major exoproteases have been used extensively in studies and applications of the secretion of heterologous proteins (Table1). A further decrease of proteases has been obtained by introducing deletions proof tease genes inspo mutants (1 14,132).However, the residual extracellular proteolytic activityof the mutants defective only inthe major proteases similar or even lower levelscan also be achieved is not particularly low, and in mutants constructed with random mutagenesis (64,85). The residual protease activityafter elimination of the major proteases was believed to be due to minor exoproteases (see, e.g., Ref.109). Indeed, up to five such proteases have been identified, with subsequent cloning

Expression Gene

in RecombinantBacillus

n

and deletionof the respective genes. There are three serine proteases: Epr extracellular protease (encoded by epr (134);bacillopeptidase F, which has also unique high esterolytic activity (encoded by the gene designated bps in Ref. 135 and bpt in Refs. 130 and 136), and another minor extravpr (137). Furthermore, there is a minor cellular serine protease encoded by metalloprotease, Mpr (138), and a novel neutral protease, NprB (1 12, 139). Elimination of these proteases by deletion mutations decreased the level of exoprotease activity,but the removal of any single gene doesso only due to the commoderately (134,137,140).The modest effect is probably pensatory increase in the amount ofother minor exoproteases, someof them possibly still to be identified (112,137).Deletion of one protease may either increasethe expression of the remaining protease genes, or a new proteolytic activityis provided by a protease, which is degraded by a deleted exoprotease. For example, deletion of bpr caused about a 20fold increase in extracellular Mpr (1 35). Multiple deletions of exoprotease genes invarious combinations reduced the exoprotease level significantly below 1% of the wild-type level(1 12,137,141).However, eventhe simulsix exoprotease genes left some active residual protaneous deletion of the tease, presumably due to some unidentified protease(s). Releaseof some intracellular proteases may also contribute to the residual extracellular protease activity;the deletion of the gene for the major intracellular serine protease, ips (142),has therefore been combined with exoprotease deficiency in many strains. Studies on model proteins have demonstratedthat the use of exoproteasedeficient host strains improves both the yield and stability of secreted heterologous proteins. The most widely used model protein with very representative results in such studies has the been TEM P-lactamaseof E. coli. Its peak level, expressed from different secretion vectors, increased severalfold and its degradation in late stationary cultures decreased dramatically when the production host lacked the two major proteases (apr and npr genes) (e.g., Refs 85 and 118). Further improvement in stability was achieved by using the strain deficient in six exoproteases (AnprE, AaprE, Aepr, Abpf, Ampr, AnprB);a secretion vector basedon theP43 promoter and the signal sequence ofthe levansucrase was usedin the studies, in the presence of multiple copies ofdegQ (see also the section above) (1 12). Comparable stability of a eukaryotic protein,a single-chain antibody molecule secreted by this vector system and this strain, was achieved at the level of5about mg of protein per liter of culture (1 19). The eliminationof only the two major proteases increasedthe stability only slightly compared with the produc-

78

Sanras

tion host with all wild-type exoproteases (119). The proteasedeficient strains of B. subtilis are clearly the obvious choicefor the production of most, if not all, heterologous proteins secreted B. in subtilis. As pointed above, hosts devoid of the major proteases have already been used in most of the experiments to produce eukaryotic proteins of practical interest (Table l), with favorable effectson yield and stability. There are, however, no reports in the literature that clarify whetherthe use of multiple deletion mutants could further improve the production of these proteins to the same degree as has beenfound with the model proteins.

2.7 Other Factors Influencing the Secretion Heterologous Proteins

of

There are indications that proteases may not be the only cause of poor performance of secretion vectors. In particular, the elimination of exooften left thepeak levelof the proteases downto a very low residual level secreted heterologous protein disproportionately below the optimal capacity of the secretion system, as judged by the expression of a homologous reporter protein. A high rate of transcription hasbeen found in such cases (e.g., Refs. 65, 73 and 108), strongly suggesting a hindrance in or incompatibility of the heterologous proteins with the secretion machinery itself. Schein etal. (73) addressed this directly by studying the secretion of IFNa2 with the secretion vector based on the a-amylaseB.ofamyloliquefaciens (64). Under conditions where only a minimal amountof exoproteases was produced (a low-protease host strain and early stationary phase of growth) and where no measurable degradation of interferon occurred in the growth medium, several-fold less interferon than a-amylase was secreted from the bacterium, as judged by pulse-chase labeling. Moreover, there was significant cellular accumulationof the precursor with an cleaved signal peptide. Clearly, the interferon so produced is either retarded in its passage or translocation across the cytoplasmic membrane, or it is aggregated in the cytoplasm. A cell-bound form of a poorly secreted protein has been 1, suggesting that retarreported in several of the studies shown in Table dation in the secretion of heterologous proteins inB. subtilis is not an exceptional occurrence.Very suggestive ofa block in secretion the is cellular accumulation,and harmful effecton growth, of the human natriuretic a-factor when expressed from an effective secretion vector (107). A block at a very early stage of secretion was demonstrated for,the outer membrane proteins OmpA and OmpF of E. coli. When expressed as a

Expression Gene

in Recombinant 8acillus

79

fusion to the signal peptide of the a-amylase of B. amyloliquefaciens,they accumulated intracellularly with an uncleaved signal peptide, showing no indication of engagement with the secretion machinery B. ofsubtilis (143). Translocation acrossthe cytoplasmic membraneand cleavage of the signal sequencetook place, however, when the level of expression was low (144). As such, these findingsare rather unexpected, becauseouter membrane proteins are translocated across the cytoplasmic membrane in E. are secreted effectively coli just like periplasmic proteins, most of which in Bacillus (above). A differentpattern of the blockage of secretion was found for human serum albumin (145). There was no secretion fromB. subtilis using a secretion vector based on the a-amylase geneof B. amyloliquefaciens, strain B, but low-level secretion was achieved from protoplasts after removal of the cell wall. However, even in cells with intact cell walls, there was both low-level synthesis of albuminand some cleavage of the signal peptide ofthe construction (albeit partial), indicating translocation across the cytoplasmic membrane. The cell wall maythus be a hindrance for the secretion of a heterologous protein. Interestingly,both the efficiency ofthe cleavage ofthe signal peptide and the secretion of albumin from protoplasts was inversely of difrelated to the efficiency ofthe synthesis of albumin, when vectors ferent levels of expression werestudied. Unfortunately, virtually nothing is knownabout theway even naturally secreted proteins of Bacilli traverse the cell wall after they have crossed the cytoplasmic membrane and are released from it, or on their interaction withthe components of the cell wall during the process of secretion (for a review, see Ref.146). So far, human albumin has remained the only heterologous protein, the secretion of which has been clearly demonstrated to be blocked at the level of the cell wall. However, the mode of its interaction with the cell wall has not been described. Similar problems with some other heterologous proteins might, however, be anticipated. Taken together, the foregoing findings pointto an incompatibility of varying degree between the components of the cellular secretion machinery of Bacillus and many heterologous proteins, especially those of eukaryotic origin. At present,this is a formidable problem efforts in to optimize the secretion of proteins of interest, requiring identificationof the limiting factors as well as characterization of their interaction with the secreted protein. The incompatibility could materialize at any stage of the secretion. First, the heterologous protein may be retarded during translocation through

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the cytoplasmic membrane;the few pulse-chase experiments performed, indicating slow export and processing (above), would be consistent with a hindrance at this stage. However, very little is known about this process at the molecular level inBacillus. As discussed above, not even all of the cellular componentsof the secretion machinery have been identified, and any studies on specific interactions of the machinery with the secreted protein itself are seriously hampered Bacillus in by the lackof an in vitro assay for translocation. Second, the precursorof the secreted protein. has to be folded properly and maintained in the conformation compatible for export before the step of translocation. As pointed out in previous sections, this involves the interaction of several chaperonesand the recently characterized bacterial signal recognition particle. Our understanding of the specificity of these factors is poor. The pattern of intracellular precipitation inan expressiondependent manner is indeed very suggestive ofinappropriate folding due to the lack of interaction with a chaperone. At least one chaperone, the SecB proteins of E. coli, is known to be specificfor a defined set of exthat incompaported proteins (fora review, see Ref.26). It seems possible tibility with bacillar chaperones could be a common reason for the poor export of heterologous proteins.This also suggests that the effect of introducing new chaperonesto the production hostto improve secretion should be explored.

2.8 Influence of Junction Between the Signal Peptide and the Mature Region Much effort has gone into studying the optimal way in which the signal sequence of the secretion vector shouldbe joined to the N-terminal end of the protein to be produced. A major role of the signal peptide isto keep the precursor ofthe secreted protein in a loose, export-compatible conformation, by interacting with the mature part of the exported protein. Mutation studies showthat a mismatch betweenthe signal peptideand the Nterminal portionof the mature region can affect the efficiency of secretion by introducing fast, tight folding of the precursor(147); and optimal amino acid sequenceand/or conformation at the junctionbetween the signal peptide and the heterologous protein can be anticipated to favor secretion. A further constraint on thenature of the junction is the need to preserve the cleavage site ofthe signal peptidase, with astructure facilitating the

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the optimalrate of translocation and release of the protein from the cytoplasmic membrane. For many but not all applications, an additional goal is the release of the protein in a mannerthat generates a product with a homogeneous, native-like N-terminus. Direct fusionof the last amino acid residue of the signal peptideto the N-terminal residue ofthe protein of interest has usually resulted in both secretion of the protein and cleavage of the signal peptide in all the secretion systems described. However, it should also be noted that the cleavage may be blocked or severely hindered by positively charged residues immediately downstream fromthe signal peptide; such residues are found of some eukaryotic proteins(148). In among the first N-terminal residues a pattern consistent with the cleavage of signal peptides of natural proteins (148,149) the specificity of the cleavagewas found to be largelyindependent of the residues on the side ofthe mature region. The cleavage may thus produce a product with the native N-terminus. However, this has not always been the case. A mixture of cleavage products differing in afew N-terminal amino acid residues in length has also been found. One possible reason may be proteolytic cleavage, or exoproteolytic nibbling after the correct cleavage of the signal peptide and after the release of product to the medium. In some cases, the N-terminal sequences of the product show that cleavage hasnot taken place after the signal peptide,but at sites conforming to the specificity of the signal peptidases and in the vicinity of the correct cleavage site (e.g., Refs. 68, 111 and 150). This is in agreement with the experimental evidence that the length of the C-terminal domain of bacillar signal peptides canbe varied with little influence on the efficiency ofthe cleavage of the signal peptide. The change in length can redirect the cleavage to a nearby site with a proper sequence(79,127,148). Accordingly, it may be reasonable to modify the junction between the signal peptide and mature region to contain only one signal peptidase cleavage site, resulting in exactly the product desired. The efficiency of the secretionthe of heterologous protein may also be affected by insertingan additional peptide between the signal peptide and the protein to be produced, often the consequence of introducinga (multiple) cloning site in the secretion vector. A systematic analysis of the effect of relatively long peptides of "random" sequence 20 to 70 (amino acid residues) between the signal peptideof the a-amylase of B. subtilis and the reporter protein, a-amylase of B. licheniformis, showed moderate

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decrease, drastic decrease, or moderate (about 1.5-fold) increaseof the secretion, depending on the particular peptide(79). In another study, the structure of the signal/mature junctionwas varied by extendingthe signal sequence by up to five codons of the mature region of the a-amylase (the protein on which the vector was based on), using additional codons derived from the cloning site constructions (70,151). The maximal level of secretion ofthe reporter proteinswas achieved with constructions of about four intervening amino acid residues; the length of the peptide,but not its sequence, seemedto be the crucial determinant. The level of secretion was about twofold in comparison with a nearly “direct” fusion; other insertions decreased secretion down to 50%. In agreement with these findings, short peptides derivedfrom a cloning-site oligonucleotide nextto the signal sequence have turned out in other secretion vector constructions eithernot to affect secretion or to decrease it moderately. Interestingly, extendingthe secretion signal inthe secretion vector to include even significant segments of the N-terminal region of the appropriate mature secretory protein has not improved the performance ofthe vector nor decreased secretion, although such constructions would be expected to contain an ideal signal peptidase cleavage siteand possibly optimize the match between the signal peptide and the (fusion) protein to be produced (12,64,9!3,101). One more strategyto optimize the region isto construct a hybrid secretion signal, where the N-terminal part of the signal sequence derives from the gene ofthe promoter of the secretion vectorand the C-terminal part from the gene of the protein to be produced, extending directlyto the mature region of that protein. The rationale for this construction is to create mRNA with a native-like 5‘ part in order to optimize expression, and as native a fit as possible with the signal peptideand the mature region of the protein. Indeed, such a construction was up to fourfold more effective than direct fusion when tested with a periplasmic galacturonase and using the secretion vector based on the a-amylase of B. amyloliquefaciem (1 52). One can conclude that there is considerable gainto be achieved bytuning the amino acid sequence at or around the junction between the signal peptide ofthe expression systemand the N-terminus ofthe secreted, heterologous protein. However, there is clearly no single optimal strategy or structure, and the optimization remains an empirical task; none of the strategies discussed have been reported to have been applied to more than one model protein on a comparative basis.

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2.9 Bacillus brevis as a Host for the Production of Secreted Protein An original approach to the design ofan efficent host-vector system devoid of some of the constraints of B. subtilis has beenthe development of B. brevis for the secretion of heterologous proteins. This has been a longterm pursuit inthe laboratory of S. Udaga, alsoreviewed recently bythe same group(153,154). The host systemis based on two B. brevis strains as 47 (155)and HPD31(156). These isolated by this laboratory, designated strains have a cell-wallstructure with unique features. Onthe surface of cell there is a layer of two proteins in an hexagonal lattice, each about100 kDa ( M W P , middle cell-wallprotein, and OWP, outer cell-wall protein). In an unusual way, these proteins are shed fromthe cell inthe stationary phase of culture. However, the synthesis of the cell-wall proteins continues and they are secreted to the culture medium for hours, resulting in high extracellular concentrations, 10 and 20 g/L in laboratory media in the strains47 and HPD31, respectively. This indicates that this B. brevis system has a high intrinsic capacity for protein secretion and that the genes ofthe cell-wall proteins are highly active. The cell-wall proteins are encoded bya two-cistron operon (157,158), whose promoter region5'-and cistron secretion signals (encoding M P ) are also obvious candidatesfor the construction of an expression vector. A further major attraction of the B. brevis system isthe very lowor nearly nonexistent level of extracellular proteases. In late-stationary-phase cultures, exoprotease activity of 1.6% (strain 47) and less than 0.02% (MPD31) of the amounts in the culture supernatant of B. subtilis have been reported (159). This suggests that B. brevis could be an exellent alternative host to exoproteasedeficient mutantsof B. subtilis. However, data on the stability of notoriously protease-sensitive eukaryotic proteins in the system are not yet available. A set of secretion vectors with the promoter region (158)and the signal sequence ofthe 5' proximal geneof the operon (the MWP) have been described. The plasmids used are either derivatives ofthe PUB1 10or of an endogenous high-copy-number plasmid of B. brevis (160). Initially, the capacity of the system was studied,and demonstrated with exoproteins from relatedBacillus species. These include thermostabile a-amylases of B. licheniformis and B. stearothermophilus, both secreted at very low levels by the respective species and both of obvious industrial interest, and an esterase of B. stearothermophilus and the sphingomyelinase ofB. cereus (156,161). Allof these enzymes were indeed expressed and secreted,

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with proper cleavage of the signal peptide. The maximal level of synthesis varied from0.1 to 2 g/L of the protein of interest in the culture supernatant; the highest values were obtained with the strain HPD31 in a very rich medium. The amounts of these proteins at arethe level of industrial interest, although theyare one order of magnitude belowthe yields ofthe secreted cell-wall proteins themselves. The feasibility ofthe system for the productionof eukaryotic proteins has since been demonstrated. However, not unlike in the case ofB. subtilis secretion system, the production was much lessefficient than for Bacillus proteins. The best results were obtained with human epidermal growth factor (hEGF), with the partof the gene encoding the mature protein fused directly to the signal sequence of the M W gene. Upto 250 mg/L of hEGF was secreted under optimal conditionsand after 3 days of growth (159) (see also Section2.4). The productwas a major protein of the culture supernatant, although the amount of contaminating MWPand O W proteins concomitantly secretedby the host were not measured. In agreement with the virtual lack of proteolytic activity of this strain, there was no evidence of degradation of hEGF. The amountof hEGF compares favorably with the best yields obtained with B. subtilis secretion systems, with spectacular stability of the product. Regrettably, such high yields were not obtained with other eukaryotic proteins of higher molecular weight, although their secretion was shown to be feasible. Usinga noncharacterized mutantof strain 47 with enhanced production capacity,60 mg/L of human salivary a-amylase was secreted. Swine pepsinogen, 10 mg/L, was secreted in native-like conformation and was activated autocatalytically under acidic conditions(see Table 1 for references). TheB. brevis system is clearly an elegant and interesting approach with much potential. But studies on the B. brevis system also showed that the poor compatibility of eukaryotic proteins with the secretion machinery inBacillus is not limitedto B. subtilis or B. Iicheniformis hosts. The extremely high levels of cell-wall proteins secreted concomitantly withthe produced proteininto the growth medium inthe B. brevis system complicate purification; secretion of cell wall proteins may also compete withthat of the protein of interest. One obvious step for further development of this system would be deletion ofthe chromosomal MWP and OWP genes. 3 CONTROLLEDGENEEXPRESSION

Despite their inherent efficiency, production systems based on the a-amylases and exoprotease genes of Bacilli have undesirable properties. As

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pointed out above, protease genes are naturally active onlyafter the exponential phaseof growth, when there also are rapidly increasing amounts of exoproteases in the culture medium. This problem is avoided in the case of a-amylase-based vectors; the expression signals of the cloned amy gene ofB. amyloliquefaciens are active in both the logarithmic and stationary phases of growth (e.g., Ref. 65). Gene of a-amylasesand the exoproteases are under catabolic repression and repressed by glucose, see above for the abocomplicating the use of very rich media. (However, lition of the glucose repression of the secretion vector based on the a-amylase of B. amyloliquefaciens, and Ref. 71) Glucose in the medium therefore cannotbe used to repress the initiation of sporulation, a convenient way to decrease the amount of exoproteases. Finally, the expression of a-amylases and exoproteases is not inducible in away practical either under laboratory conditions or in large-scale applications. This excludesthe possibility of using these expression signals for products, which,at a high level ofproduction are harmful to the cell, but whose production would be feasible ifthe expression of the gene could be limited to a short production phase at high cell density. Such a production mode could also increase the stability of some expression plasmid constructions wherethe very synthesis ofthe protein to be produced may contribute to the structural instability of the plasmid.On the other hand, it should be pointedout that secretory expression as such is oneway to avoid harmful effects that may be caused by the intracellular accumulation of the product. The inducibility of secretory production as such may result only in a modest improvement in the process. A protein effectively secreted is not expectedto be harmful to the cell. Furthermore, the typical mode of expression in an inducible system is ashort period of production at a high cell densityand at a very high level of expression. This is not feasible with secretory production, becausethe maximal capacity of cellular secretion isthe rate-limiting factor in all present applications. The feasibilityand usefulness of inducible expression have been amply demonstrated by the multitude of such production systems developed and routinely available for E. coli. Development along similar lines has now resulted also inthe availability of bacillar expression systems with a more favorable mode of expression than those basedon a-amylases or exoprotease genes. There are already several vector systemsfor inducible synthesis basedon various homologousand heterologous genes and genetic elements. As described below,the majority of these have been developed with the initial or explicit goal to construct a secretion vector system with improved properties; they have been mainly studied and characterized with such applications in mind. On the other hand, many of them canor

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have been easily modified for the expression of heterologous genes for other purposes.

3.1 Expressionwith Bacillar GeneticElements The shift to the exponential phase of growthand insensitivity to glucose was achieved bya constructionof Wong et al. (1 18). A strong vegetativephase promoter froman unidentified gene ofB. subtilis was cloned, characterized, and fused to the ribosomal binding siteand signal sequenceof the subtilisin gene B. ofsubtilis in aPUB1 10-derived plasmid. When tested with a reporter gene (the TEM 0-lactamaseof E. coli), a good level of expression and secretion was achieved. The combination of a very rich of medium containing3% of glucose further increased the amount secreted 0-lactamase, rendering it stable without any degradation, even during extended timeof incubation (upto 100 h), with a high density of cells in the culture and without sporulation. A very different principle has been applied in the laboratory D. Behnke A production to achieve expression in the exponential phase (1 14,129). system based onthe heterologous gene(sak)of bacteriophage 42D of S. aureus encoding the secreted staphylokinasewas developed. This gene is active inB. subti&, where it is expressed constitutively throughout growth (1 14,129).The protein is secreted efficiently inB. subtilis. A “portable” expression/secretion signalwas constructed by fusing a fragmentof about 400 bp containing the promoter and the signal sequenceof the staphylokinase geneand a polylinkerto the end of the fragment in a set of hybrid plasmids derived originally from Streptococci. The applicability of the production system was demonstrated with the gene of human interferon a1 (IFN-el). In a proteasedeficient B. subtilis strain, there was secretion of IFN-al, beginning in the expotential phase of growthand extending constitutively to the stationary phase; activity of the promoter was also evident inthe late stationary phase. The sak expression system thus shows flexibility for different modesof production. However,the peak level for I F N was only about 15 mg/L of culture with indications of proteolytic degradation. Thereare no data for any well-expressedor secreted protein produced with the aid of this vector. One way to achieve an inducible secretion and expression system has been to start with a gene that is readily inducible. Among few secreted proteins of Bacilli with inducible expression,the levan sucraseof B. subtilis is the one best characterized. The synthesis and secretion of levan sucrase are induced by exogenous sucrose, also in the exponential phase

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of growth (162,163). The activityof the correspondingsacB gene is regulated by several well-characterized regulatory mechanisms and genetic elements, which can be utilized inthe construction of a secretion system not only to achieve inducibilitybut also to further enhance and control the production. A region calledsacR between the promoter of the gene and sacB (the structural gene of levan sucrase) contains a transcription terminator (a stem-loop structure), which ismodulated by sucrose via the function of the product of sacY (in sacs locus), involving its presumed phosphorylation and binding to sacR, thus providing antitermination and induction of sacB (164,165). Further control is provided bythe set of the pleiotropic, mainly positive regulatory genes, which affect the activity of a number of genes encoding secreted degradative enzymes. These include degQ, degU, and prtT. The well-characterized targets of these regulatory systems are upstream of the sacR region (89,162,166). Expression systems utilizing these features have been constructed in several laboratories. The Rapoport group (108,110,162) has used sacRB the region of B. subtilis to construct inducible secretion vectors based on the multicopy plasmid pC194. The expression signals were in a DNA fragment of about 550 bp, which contains the sacR region, the promoterof sa&, and the complete signal sequenceof the levan sucrase. DNA fragments encoding the protein to be secreted have been fused to the 3’ end of the fragment, providedby unique cloning sites either for the exact fusion of the DNA fragment of the “N-terminal” codonto the cleavage site of the signal sequence, or by inserting afew amino acid residues between the signal peptide and the N-terminus. The secretion of three heterologous proteins with the aid of this basic construction has been reported. The desired sucrose-dependent expression and secretion were initially demonstrated with the TEM 0-lactamase as a reporter; the sacs locus was the one in the chromosome. Although the amountof 0-lactamase secretedwas described only in arbitrary units, the presence of inducibility was shown bythe absence of P-lactamase activity in cultures without sucroseand a steady increaseof activity in the exponential phase of culture in the presence of sucrose (162). It should be noted that although thesacR region was present in multiple copies, there was only one chromosomal copy sacY/sacS; of this imbalance did notseem to interfere with induction. More quantitativedata were reported for the secretionof a bacillar exoenzyme, the cellulase ofB. thermocellum, presumed to be protease resistant (1 11). Again, therewas no detectable secretionof the proteins in un-

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induced cultures, and secretion was increased to the level of about 0.5 mg/L of cellulaseby induction in culturesof wild-typeB. subtilis in stationary phase (1 10) (but see also below). The same level of secretion was found when the signal sequence of levan sucrase was replaced by the native signal sequence ofthe cellulase. In the case of a third construction, the reporter was murine interferon-a7 (108). There was no secretion in B.subtilis, but transcriptionof IFN-a7 inducible with sucrose was demonstrated. Wong (109) has constructed another secretion vector based on sacB. He cloneda slightly modified segment about of 600 bp of B. subtilis containing the upstream sequences from the 3’ end of the signal sequence of sacB as a cassette ina Bluescribe plasmid inE. coli. The matureTEM plactamase ofE. coli was then fusedto the end of the signal sequence and the construction inserted in a derivative of the multicopy plasmid PUB1 10 to study the expression inB. subtilis. A rapid increase in P-lactamase secreation was seen following the addition of 2% sucrose to a culture of super-rich media with3% glucose. Again, there were negligible amounts of P-lactamase in the absence of sucrose. A significant advantage of expression systems basedon sacBR is the possibility of enhancing expressionby increasing the transcriptionof the fusion genes via the activity of degQ, degU, and degS genes (formerly sacQ, sacU, and sacs, respectively). Increased levels of thedegQ regulatory protein(e.g., from multiple genes in a plasmid or due to anup-promoter mutation) or degU (Hy) and degS (Hy) mutations (for a review, see Ref. 89) increase transcription of sacB. Indeed, significant enhancement of secretion has been achieved by taking advantage of this regulatory system. Usingthe sacB secretion system above, Joliff et al. (110) demonof heterologous strated abouta six- and eight-fold increase in the secretion cellulase indegS (Hy) and degU (Hy) mutants of B. subtilis, respectively. In both mutants, the expression was still inducible by sucrose. Up to 5 mg/L of cellulase (in contrast to 0.5 mg in the wild-typestrain) was secreted in the early stationary phase when the expression cassette was ina plasmid with a copy number ofabout 20. Furthermore, increasing the copy number of the expression cassetteto the extraordinarilyhigh number of 250 (see also Section 4), by integrating it tandemly in the chromosome ina degU (Hy) mutant (Ref. 111 and Section 4), resulted in a fourfold increase in secretion relative to the plasmid system. Wong (109) has studied the feasibility of enhancing expression based on sacRB by increasing the expression of the cloneddegQ gene ofB. subtilis in the production host.He cloned degQ (sacQin 109)in asacB-based

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secretion vector expressing the P-lactamase as a reporter. The copy number of degQ was thus matched withthat of the expression signals.A high level of degQ expression was provided by placing the initial promoterless degQ gene under the strong P43 promoter.*This promoter is highly active in both the exponential and stationary phases of growth. Introduction of the degQ gene enhancedthe secretion of P-lactamase 17-fold. The author also studied the effect of degU(Hy)mutation in the host strain no addiand founda %fold increase in P-lactamase secretion. There was tional effect of degQ(Hy) in the strain carrying the plasmid with cloned P43-degQ fusion. A potential drawbackof enhancingthe production by hyperactivating any degsystem is the simultaneous increase in the secretion of exoproteases, even ina protease-deficient host. Overexpression of degQ increases the activity of secreted minor proteases inapr and npr mutants (138). It has been shownto decrease significantly the half-life E.ofcoli P-lactamase expressed from a secretion vector, even ina mutant defective in several exoprotease genes (112). However, in some secretion vector systems, as in the oneused by Wong (109) and in the secretion vector system based on the neutral proteaseof B. amyloliquefaciens (IOI), overexpression of degQ was not foundto increase the exoproteaselevel or the rate of degradation of a secreted model protein. Zukowski et al. (167,168) have constructed a sacB-based inducible expression vector explicitly for intracellular protein production B. in subtilis. The vector,a derivative of pC194, hasan insert comprising either the native sacR regionor the fusionof a synthetic bacteriophage T5 promoter with truncated sacR region (containingthe stem-loop regionbut not the promoter). The T5 promoter, although of gram-negative origin by nature, is well recognized inB. subtilis and, when fusedto a promoterless reporter gene (chloramphenicol acetyl transferase of S. aureus), can mediate very efficient synthesis of the respective protein to(up 25% of total cellular proteins) (169). TheVIE gene-of Pseudomonaputida (encoding intracellular catechol 2,3-dixoygenase), with its ribosomal binding site, was by a fragment fused to the 3’ end of the truncated sacR region, followed containing the transcription termination siteaof phage ofE. coli. Expression of the reporter proteinwas moderately inducible by sucrose in both cases. Slightly higher noninducedand induced levels ofthe reporter were observed whenthe expression plasmid contained an insert with the sacY/ sacs region, resulting additionally in presumed overexpression of the positive regulator sacY (the antiterminator). However, the maximal levelof expression was only10 to 20% of the very high constitutive level obtained

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from the T5 promoter in a construction without the stem-loop region of the sacR region, in agreement with the similar difference observed between the maximal sucrose inducible level of expression of sacB+ and that of a stem-loop deletion(168).

3.2 Induced Expression with lac Elements of E. coli In E. coli, inducible expression based on the P-galactosidase (IacZ)gene, the lac repressor (lacl), and induction by IPTG (isopropyl-o-thiogalactoside) has been the basis of a number of practical and widely used expression systems. These elements have also been introduced to control gene expression in Bacillus in several systems. Indeed, the earliest inducible expression of heterologous proteins inB. subtilis was based on this principle in the laboratory of D. Henner, as described below. Yansura and Henner (170) constructed two hybrid expression signals based on bacillar promoters, designatedPpac-Z and Pspac-I, and fused to a lac operator sequence (a sequence of 35 bp containing the target of site lac repressor), followed downstream by a cloning site for the gene to be expressed. This cassettewas combined withthe lacZ gene brought under the control of an active Bacillus promoter, the one from the B. licheniformis 0-lactamase gene(blaP).The pacZ promoter isa derivative of the foregoing blaP promoter, comprising a fragment of about 100 bp with the lac operator sequence, situated between the - 10 region and ribosomalbinding site(RBS) of blaP. The spac-Z promoter is composed of a fragof the B. subtilis bacterioment of about 200 bp from the promoter region phage SPOl, the lac operator sequence,and a synthetic ribosome binding site fused downstream ofthe - 10 region, with cloning sites between the RBS and the initiation codon at the 3' end of the fragment. The expression vectors described in Ref. 171 containedtheinsame vector thePpacZ or Spac-Z cassette and thelacZ cassette; the vector was an E. coli-B. subtil& shuttle vector. The functionality of the mac-Z was demonstrated with the P-lactamase ofB. licheniformis and that of SpacZ with human interferon-a. In bothcases there was a 50- to 100-fold difference in enzymatic or biological activity inthe presence and absence of IPTG in the growth medium, respectively. et al. (172) to inThe lac operator and lacZ were also used by Le Grice troduce inducibilityinto a promoter, which was modified from theE. coli bacteriophage T4 promoter, lac operator and a RBS derived from phage T5, in an E. coli-B. subtilis shuttle vector. The IacZgenewas introduced into theB. subtilis production strain ina second multicopy plasmid under

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the control of a promoter adapted for expression in B. subtilis. Expression in this system was also shownto be dependent on IPTG induction (172); synthesis ofthe protein of interest couldbe induced inthe late exponential phase for efficient production (173). Interestingly, the hybrid tac promoter of E. coli, derived from the lac and try promoters, is also weakly active inB. subtilis (169) and repressed by lacI(174). The promoter was inducible byIPTG; due to the low level of expression,the repressibility was high, suitable for cloning genes with detrimental expression (174). 3.3 Temperature-DependentControl Rapid induction ofthe synthesis ofthe protein to be producedby a temperature shift might be advantageous in many systems, compared with induction by chemical agents, although a definedlevel of production is more difficultto achieve and the high temperature may be harmful to some proteins of interest. Two systems, both analogousto the widely applied thermoinducible systems of E. coli, based on temperature-sensitive repression of lambda phage and the appropriately controlled promoter, have been described for B. subtilis. Two laboratories have constructed dual vector systems with the expression signals of an early promoter of the B. subtilis phage $105. In both systems, one plasmid containsthe native promoter and operator of this phage gene fusedto the heterologous geneto be expressed. Inthe second plasmid, pE194,there was an insert containing the represser gene of this early promoter. pE194 is nonreplicating at the restrictive temperature of 45°C (175,176).The repressor gene described in Ref. 175 is wild type, and temperature induction of the promoter is achieved by nonreplication the of plasmid after the shift to 45"C, with subsequent slow diluting out of the repressor in the course of further growth. In the second system the repression gene encodes a temperature-sensitive mutant repressor (176); more rapid inductionof the synthesis by the promoter at the temperature shift is anticipated. Upto a 20-fold induction ofthe synthesisof a reporter prois relatein was obtained in the latter system. However, the 6105 promoter tively weak and thesystem as such is not suitablefor high-level production. Breitling et al. introduced the elements of the temperature-sensitive induction of theE. coli X phage system inB. subtilis (177). They constructed an expression plasmid containing two inserts: one, the expression cassette, contains theP, promoter of the X phage with its upstream region, the second insert contains the temperature-sensitive X C1857 repressor, brought under thecontrol of the sak42D expression cassette of S. aureus

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origin (see above,and Ref. 129). The plasmid is a multicopy plasmid of streptococcal origin. The system has been tested using the secreted a-amylase of B. amyloliquefaciens as a reporter, the gene being linked to the end of the expression cassette with its signal sequence. Impressive activation of a-amylase secretion greater than 1400-fold was achieved with a temperature shift from 30°C to 42"C, althoughthe maximal level of secretion was only modest.

4 CHROMOSOMAL EXPRESSION SYSTEMS Expression systems based on the use of plasmids are not an optimal choice for large-scale applications because they are inherently unstable. Plasmids can be lost by segregational instability without selection pressure. Moreover, there can be structural alteration during extended growth, with concomitant lossof the activity of the gene to be expressed. Such changes may be favored if the protein to be produced affects the growthof the cell. Plasmid instability isan annoying problem, especially with the Bacillus cloning and expression vectors. Those most widely used are derived from S. aureus, and due to their mode of replication, are especially liable to a high level of plasmid instability (3). The maintenance of plasmids with the prevailing method,that is, by using antibiotics for selection, is undesirable in industrial applications because of the high cost involved and for the possible environmental and medical risks that can result from increasing the prevalenceof antibiotic resistance in microorganisms. The maintenance of the plasmid inthe expression system with selection not based on antibiotics and feasible in industrial growth media has also been described (178). Integration of the heterologous expression cassette into the chromosome of the production host provides a way to stabilize its inheritance. The rate of recombination of chromosomal DNA is significantly lower than that in plasmids, greatly improving the stability of the inserted gene. There are methods availablefor the tandem duplication of the integrated gene to achieve high levels of production.A number of integration vectors have been developed and described for the purpose. Theseare (usually) plasmids of E. coli, amended with a (antibiotic) resistance gene active in B. subtilis and with a segment of DNA homologous to a region of the chromosome but devoid ofa replicator activein B. subtilis. An exemplary and widely usedconstruction inthe plasmid pHJ101, a derivative of pBR322 with a chloramphenicol resistance gene (from the S. aureus plasmid pC194) conferring resistance inB. subtilis and E. coli (179). If provided withan

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insert containing sequence homology with chromosomal DNA,it can confer chloramphenicol resistance when transferred to B. subtilis by integration of the entire plasmid in the chromosome at the site of homology. The integration takes place by Campbell-type recombinationat high frequency by one crossing-over event. The same principle has been applied using a number of derivatives of E. coli plasmids containingdifferent antibiotic markers selectable B. in subtilis (86,111,145,180,181). Homology with the chromosome can be provided by a chromosal fragment in the plasmid as above,or by preinsertion of heterologous DNA present in the integration plasmid into the chromosome of the B. subtilis host strains (181-183).

A crucial consequence of Campbell-type integration isthe generation of of directly repeatedDNA sequences flanking the inserted segment DNA. Such segmentsare inherently proneto multiplication or amplification in the course of replication of the chromosome. Amplified structures can readily and conveniently be selected, if, as usual, theantibioticresistance gene in the integrated plasmid shows a gene dosage effect and thus amplification of the structure confers resistance to higher concentrations of the respective drug. Amplification of inserted segment DNA of can readily be achieved by the (gradual) selection of increased resistance. Typically, upto 10 to 30 copies of the amplifiable segment have been obtained by straightforward selection for chloramphenicol, kanamycin, or erythromycin resistance conferred by the respective genes in the plasmid. Furthermore, a desired level of amplification canbe designed and maintained to a certain degreeby adjusting the concentrationof antibiotic in to adjust and reguthe growth medium(181,184). This makes it possible late thelevel of synthesis ofthe protein to be produced bythe choice of a production strain with the appropriate level of amplication of the expression cassette. The highest level of amplificationthat can be obtained dependsboth on the antibiotic used, and in a way not yet clarified, on the repeated sequence flankingthe structure (181,182). An antibiotic resistance geneexpressed through a relatively weak promoter will provide favorable conditions for a high level of amplification selectedfor resistance. Multiple copies of an integration plasmid have also been introduced into the-chromosome by multiple integration steps, resulting ainset of strains witha well-defined and well-controlled integrant copy number (185). Integrated and amplified heterologous structures in the chromosome of B. subtilis can be very stable, although not invariably. Virtually no loss

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of or decrease in the level of amplification has been observed during the 30 to 100 generations of growth without selection pressure in the case of various integrated structures, even with copy numberstoup30 (181,183, 186). This was still true when the integrated and amplified plasmid (copy number 5 to 9) encoded a protein through an effectively expressed promoter; there wasno detectable loss of the vector ina 40-h batch culture (185). On the other hand, some integrated structures, possibly depending on the flanking, repeated segment, are less stable and may eventually be lost without selection pressure (185). The stability may also be affected unfavorably by a high level of expression ofthe heterologous gene in the construct (182). A molecule of DNA containing heterologous sequences can also be integrated into the chromosomeas a consequence of two crossover events, each in one of two regions homologous with the chromosome and flanking the heterologous sequence. The integrated molecule replaces the chromosomal segment between the regions of homology,and a stable structure is formed if the integration does not replaceor inactivate an essential gene (187,188). As in Campbell-type recombination, the moleculeto be integrated is usually introduced into B. subtilis as a part of a nonreplicating plasmid containing a selectable marker in the segment inserted. For a more detailed description of the methods of plasmid transformation in Bacillus, see for example, Ref. 189. Amplification of the inserted fragment can also be achieved readily under selective conditions (e.g., for antibiotic resistance) if the insert is constructed to contain sequences of direct repeatsat both ends. Both the levels of amplification and the stability of the amplified structures are comparable to those obtained witha single crossover(181). On the other hand, becauseno repeated sequences are generated by integration with double crossover, a fragment devoid of repeats is not amplified,a property utilized for the stable single-copy integration of heterologous genes, as 189). well as a methodof mutagenesis andan aid in cloning (see, e.g., Ref. A third method of chromosomal integration utilizes the staphylococcal plasmid pE194, which has a temperature-sensitive replicator(3). At permissive temperatures, it replicates inB. subtilis as an autonomous plasmid, but the plasmid also integrates in the chromosome at multiple sites without any reciprocal sequence homology. At elevated temperatures (above 45"C), such integrants can be selected for erythromycin resistance conferred by the integrated plasmid, which isno longer replicating autonomously. Stable strains with multiple integrationa nontandem in fashion of can be the plasmid and any heterologous genes inserted into it constructed

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(3). pE194 derivativesare also a convenient alternativefor nonreplicating plasmids whenthe goal is integrationat a site of homology, by eitherthe Campbell-type or double-crossover mode. The plasmid is initially transferred to the host at the permissive temperature. Dueto its autonomous replication, transformantswith a high plasmid copy numberare obtained even if the effectiveness of the transfer is low, as in electroporation. A subsequent shift to a nonpermissive temperature under antibiotic pressure then integrates the plasmid into the chromosomeat a high frequency, with permanent loss of the autonomous plasmid. Several expression systemsfor heterologous proteins basedon expression cassettes integrated in the chromosome have been described. Saunders et al. (186) and Fahnestock et al. (180) deliberately constructed strains with a very low level ofamplification (from 1 to 2-4 copies) of staphylococcal genes encoding secreted P-lactamase and protein A, respectively. The constructions were stable and the proteins were synthesized and secreted at high levels in the case of proteinA. Both studies demonstrated that the possibility of constructing an expression system with a stable low copy number of the expression cassette may be very useful. It was not possible to express either gene from a multicopy plasmid, presumably due to the harmful consequences to the cell resulting from hyperexpression. Both human albumin and dihydrofolatereductase have been synthesized inB. subtilis with the aidof an expression cassette in the chromosome, human albumin from the expression and secretion signalsof aamylase and neutral protease genes of B. amyloliquefaciens (145), and dihydrofolate reductasethrough the cat promoter of the plasmid pc194 (183). Kallio et al. (86,185) compared the expression of the a-amylase gene of B. amyloliquefaciens in B. subtilis when integrated ina stable manner in multiple random sites from twoto eight copies, or carried by pUBllO the plasmid witha copy number ofabout 50 per chromosome. Interestingly, both the secretion of a-amylase and the level oftranscription in thestrain with two copies nearly equaled those of the strain carrying the plasmid construction. A further threefold increasewas obtained by increasingthe number of chromosomally integrated genesto eight.

5 PRODUCTION OF HETEROLOGOUSPROTEINS INTRACELLULARY IN B. SUBTlLlS Most efforts to produce heterologous proteins Bacilli in have focused on the extracellular mode of production, as described above. However, in

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many respects bacilli would compare favorably with alternative production hosts, even if synthesis of the protein were to take place intracellularly. Of particular importanceare the extensive experience with Bacillus in large-scale industrial applications,and its nonpathogenicity and nontoxicity, which greatly facilitates downstream processing. The main problems hampering the production of heterologous proteins in secreted form are inherent in that mode of production and may be expected to be circumvented if the proteins remain intracellular.As pointed out in Section 3, the expression systems developedfor the secretion of heterologous proteins could be applied, with simple modifications, to the intracellular mode of expression. Moreover, if high levels of intracellular production were to be achieved, resulting in inclusion bodies as with many proteins produced in E. coli, it would compensateto a degree for the lack of secretion by providing an easy first stepof purification. The feasibility ofthat approach has, indeed, been demonstrated in a number of studies. High-level productionof heterologous intracellular proteins B. in subtilis was first reported by Zukowski and Miller (167) for a reporter protein, the catechol 2,3-dioxygenase of Pseudomonas putida. Expression of the protein was from the xylE gene, with its own RBS, fused alternatively to one of three B. subtilis promoters. The gene constructions were cloned in a multicopy plasmid. A very high level of expression ofVIE was obtained with two ofthe promoters, the sacR promoter of the levansucrose gene in adeguh (designed sac@ in Ref. 167) mutant of B. subtilis and with a synthetic pro(see above) moter, with a consensus sequence of bacteriophage T5 promoter in a wild-type strain. In the deguh mutant, up to 25% of the soiuble cellular protein was estimated to be catechol 2,3-dioxydenase protein; the protein showedthe correct enzymatic activity. These data point to a considerable potential for B. subtilii as a host for the intracellular production of proteins. Intracellular production in B. subtilis was extended to a human protein of potential pharmacological interest. Wanget al. (173) have synthesized human tissue-type plasminogen activator (TPA) in B. subtilis, using a shuttle plasmid system, in which the expression ofthe heterologous protein in B. subtilis was based on an inducible hybrid promoter containing E. coli lacregulatory elementsand bacteriophage T5 promoter.TPA is a natural secretory protein, it is not compatible with the secretory machinery of B. subtilis. The stretch of a cDNA clone of TPA devoid of its signal sequence and encoding the 60-kDa mature TPA was inserted into the expression vector. IPTG-induced cultures of B. subtilis con-

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taining this expression vectorwere found to synthesize immunoreactive TPA of mature size. Estimates basedon immunoblotting indicatedthat the amount of TPA was in the range of 20 mg/L of culture, an amount clearly smallerthan thatof the catechol 2,3dideoxygenase described above, although encouraging with respectto practical applications. The reason for this comparatively small yield was not elucidated. All ofthe TPA was found in the pellet fraction of the broken cells, suggestingthat the protein was aggregated, possibly in the form of an inclusion body, despite the low level of expression (lessthan 0.5% of the mass of packed cells). However, the finding is consistent with the amounts of intracellular heterologous protein found in cases of some proteins forming inclusion bodies in E. coli (190,191). A higher levelof intracellular synthesis B. in subtilis of another human protein, interleukin-10, has been achieved by Bellirii et al. (105). Again, a semisynthetic stretch of DNA encoding the mature, secreted form of IL-10 (ca. 17 kDa) was fusedto an efficient promoter in a shuttle plasmid for expression. Analysis of the total cellular protein of the production strain indicated the presence of immunoreactive IL-10 in amountsabout of 5% of the total cellular protein. However, despite this relatively highintracellular concentration, all of the IL-1P was present in the soluble fractions of the cell. Furthermore, the biological activity of the proteinwas indistinguishable fromthat of authentic IL-10. It is thus likely that unlike TPA, the protein formed its native conformation in the intracellular environment of B. subtilis. The authors also described a purification scheme from high-density cultures of B. subtilis with yields of 35 mg of highly purified active IL-10 from 1 Lof culture. The possibility of replacing secretion with the intracellular expression of heterologous proteins has been studied extensively and applied to two types of potential vaccine proteins of gram-negative bacteria, the pertussis toxin fromBordetellapertussis,the agentof whooping cough, and a number of outer-membrane proteins, including one fromNeisseria rneningitidis, the causative agent of bacterial meningitis and other invasive infections. Pertussis toxin is a multimeric protein encodedby a five-cistron operon (192). Each subunit is predicted to be a secreted periplasmic protein by nature and synthesizedas a precursor with a cleavable signal peptide. However, only the subunit S1 was secreted effectively inB. subtilis (59) (Table l). Only insignificantamounts of subunits S2 to S4 were found extracellularly, with low intracellular accumulation of the precursors, indicating poor compatibility with the export mechanism ofB. subtilis (59). To test the intracellular expressionof these proteins, the secretion vector based

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on the a-amylase of B. amyloliquefaciens(64) was modified bytruncating most of the signal sequence ofthe expression cassette ofthe plasmid. However, the codons of the seven N-terminal amino acid residues were preserved; this stretchof nucleotides was found to be essential for expression of the protein (193). All pertussis toxin subunits (devoid of their native signal sequence) were expressed from this modified vector. Consistent with the efficiency of the a-amylase promoter, remarkably highlevels of all subunits exceptS1 were produced. The highest level achieved (for the subunit S4) was upto 500 mg/L and about 20% of the total cellular protein in shake flask cultures,and up to 3000 mg/L in batch cultures in a fermentor (194). There was no indication of degradation even during extended periodsof growth, althoughthe optimal yield was achieveda few hours after the exponential phaseof growth underlaboratory conditions. Consistent with the lack of the signal sequence inthe expression vector, all subunits were tracellular in B. subtilis, presumably inthe form of inclusion bodies. The expresion vector system described above has also been to study used the intracellular productionof a setof outer-membrane proteinsof gramnegative bacteria, includingboth model proteins,the major outer-membrane proteins OmpF and OmpA of E. coli, and a prospective vaccine antigen, the classI or P1 protein of N. meningitidis, a major protein of that organism. As described above, these proteins were synthesized at low levels and not secreted inattempts to produce them with a secretion vector system inB. subtilis, although theyare proteins normally exported across the cytoplasmic membrane in their native host. theOn other hand, . being integral membrane proteins embedded extensively in the lipid membrane in their native environment, theyare very different from the periplasmic pertussis toxin or the soluble, eukaryotic TPA. Expression of these three outer-membrane proteins from the a-amylase promoter in the vector described above produced consistent results, very similar to those obtained with the pertussis toxin subunits. Again, each 20% of the total protein was produced at a high level, in the range ofto10 cellular protein, and all were cell bound (194,195,197).It seems remarkable that this high concentration of intracellular heterologous protein was not significantly harmful to the cell. The growthrate of the strains producing outer-membrane proteins,as well as those producing the pertussis toxin subunits, was unaltered. However, moderately increased plasmid instability was indicated(M. Nurminen, personal communication).This suggests that growth had a slight effect on plasmid stability, in contrast to the high (63). stability of the variant of the plasmid expressing secreted a-amylase

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The pertussis toxin subunits and the three outer-membrane proteins were insoluble inB. subtilis, being found in the fast sedimenting fraction after breakage ofcells (193-195), consistent with the presence of inclusion bodies. Intracellular globules of electron-dense material were seen in cells expressing each of the outer-membrane proteins (194,195); these structures were very similarto the inclusion bodies observed inE. coli (196), and left no doubt of their presence in B. subtilis. The productionof a heterologous proteinas an inclusion body provides of the advantageof an easy initial isolation step, using simple procedures cellular breakage and low-speed centrifugation, as amply demonstrated with numerous proteins in E. coli (196). Using this procedure, the pertussis toxin subunits and the outer-membrane protein inclusion bodies could be isolated easily in gram amounts on laboratory a scale (193), with considerable enrichmentof the protein of interest (193-195,197). Isolation by centrifugation and enrichment was also achieved in the case of intracellular aggregatesof TPA, although the intracellular concentration was relatively low (173). The intracellular mode of production of heterologous proteins in B. subtilis can thus be highly efficient, makingB. subtilis competitive with E. coli as a hostfor the productionof a wide variety of proteins. The high initial yield,the ease of isolation ofthe inclusion bodies,and the proven industrial fermentation technologyof B. subtilis make this approach attractive, even for the production of low-cost bulk products. A potential drawback of producing proteins as intracellular inclusion bodies is, however, the formof the protein. Typically, although not invariably, the proteins are paracrystallic and in a nonnative conformation. Furthermore, they are usually solubilizable only with strong detergents orchaotrophic agents, a process leaving the protein fully denatured (190). This could discourage practical applications if the goal is a biologically active product of high purity, which can be achieved onlythrough a complex process of solubilization and renaturation of questionable efficiency. The inclusion bodies of the heterologous proteins in B. subtilis were highly insoluble and none of them were reported to be solubilized without complete de'naturation (173,193-195). However, conditionsfor effective renaturation have been developed. TPA in inclusion bodies, after solubilization and full defolding with guanidine HCL, was subsequently diluted to allow refolding. Purification with affinity chromatography then produced a protein with the native enzymatic activity of plasminogen activator (173). An effective way to renature the outer-membrane proteins produced as inclusion bodies

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has also been described; proteins solubilized with detergents or quanidine HC1 werenonnative, as judged by their immunogenic properties[PI protein of N. meningitidis (195)] or by their inabilityto bind phases [OmpA or OmpF proteins of E. coli (194)]. However, complexing these proteins with lipopolysaccharide(LPS), with the simple procedure of dialyzing the solubilizing agent away in the presence of restored the phage-binding LPS, activity of OmpA and OmpFand, very significantly, the ability of the P1 protein to elicit protective immunity an in animal model(195,197). In the case of P1 protein, LPS has subsequently been replaced with nontoxic phospholipids (Muttilainen, S., personal communication). The formation of inclusion bodies and the loss of biological activity are notalways an inevitable consequence of the heterologous intracellular expression of proteins B. in subtilis. The catechol 2,3dioxygenase of Pseudomonas, a native intracellular protein, was obviously not associated with aggregates andwas recovered as a soluble protein with full enzymatic activity in cell extracts, although produced at very high levels,as described above. Reverse transcriptase of a T-cell lymphotropic virus also retained its biological activity when produced intracellularly in B. subtilis (172) using a high-level expression system containing elements of phage T5and lac operator (169). Two human proteinsof pharmaceutical interest havealso been expressed intracellularly without loss of activity: human IFN-a2, from staphylokinase sak420 transcription and translational signals (11 9 , and human IL-lp from a strong constitutive promoter(105). The yield of human interferon, however, was low, with an indication of extensive proteolytic degradation. Indeed, the level of recovery was several orders of magnitude lower than the amount of this protein secreted inB. subtilis when (1 14). the same expression signals were used in a secretion vector system In contrast, more than 2 mg of IL-10 per gram of cells (wet weight) was found intracellularly in high-density cultures (105) and was purified at a high yield without loss of biological activity. This compares favorably with the extracellular production of the same protein, using a secretion B. subplasmid withthe expression and secretion signals of proteases of tilis; about 0.4 mg of interleukin per gramof cells was found in the culture supernatant (105). 6 PRODUCTION OF PROTEINS OF GRAM-POSITIVE BACTERIA

By and large, genes from a large variety of gram-positive species may be expressed inB. subtilis and in other Bacillus spp. from expression signals

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of their own (5,199). This applies especiallyto proteins of other bacilli, staphylococci, and streptococci, butalso to proteins of species less closely related, such as Listeria (see below). This enables the design and construction of a simple expression system, suitable for many purpose, by cloning an apspecies to a bacillar propriate large fragment of the DNA of the heterologous plasmid vector and looking for direct expression ofthe proteinof interest. Direct expression in E. subtifis can be used to produce a protein for biochemical, enzymological, structural studies often more readily than the native speciesof the protein. The native species may befastidious for growth. If the gene is clonedon a multicopy plasmid,the level of expression canoften, but not invariably,be high enoughfor ready purification, even on a large scale,due to the gene dosageeffect. Direct expression in E. subtifis of (exo)enzymes with properties of industrial interest may be the host straightforward initial step for the development of a large-scale production system. Other potential and proven applications of direct expression include dissection of the individual components of a complex system to study of their role and possibly their mode of function via their expression in E. subtilis. Interestingly, this approach has been applied successfully even as Listo the virulencefactors of pathogenic gram-positive bacteria such teria (200), suggesting a possible simplified method of analyzing other complex function of gram-positive bacteria. Expression inE. subtifiscould also provide a means to study species- or gene-specific regulatory mechanisms. All genes of bacillior other gram-positive bacteria are not expressed in E. coli, genes involved in sporulation and the development of competence among them (3). Utilization of direct expression E. in subtifis is (3). Examfacilitated bythe development of efficient direct cloning systems ples of the use of these approaches follow. Major exocellular enzymes of bacillar species used in industrial biotechnology have been expressed efficiently from expression signals of their own after shotgun cloning, in most cases on multicopy plasmids derived from pUBl10. These include a-amylaseof E. amyfofiquefaciensand E. licheniformis (61,201), and alkaline and neutral proteases of the foregoing species (87,88,202). In all these cases,the level of production was very high, comparable with or even manyfold higher (88) than that in'the donor organisms of the gene used previously in industry. subtifisof degradative bacilThere has been successful expressionE. in lar exoenzymes with propertiesof practical interest such as thermostability [a-amylases (201,203), proteases (204-207),and favorable pH range (a-amylase) (208)] or with other activities of interest [e.g., cellulases (209)

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and penicillin acylase(210)]. Many of these enzymesare secreted onlyat low levels inthe native host, and their expression inB. subtilis or other species ofBacilli for industrial production couldbe a good alternativeto traditional strain improvement with mutagenesis and other classical methods. Examples of good expressionin B. subtilis of the genes of another genus are those of streptoformase (DNAase) from Streptococcus (211) and of staphylokinase, nuclease, a-hemolysin, and P-lactamase from S. aureus (129,206,212,213). Although the method is widely applicable, some constraints forlthe direct expression of heterologous proteins from other gram-positive species can be noted. All of the cases reported to date are secretable proteins; they were also secreted in B. subtilis. This bias is clearly caused by the wide industrial interest in this class of proteins but leaves unclear whether the expression of other types of proteins (intracellular, membrane bound) might in some cases be harmful to Bacillus and thus limit the usefulness of this aproach. Some proteins were also expressed B. in subtilis at levels disproportionately low in comparison with the copy number of the cloning vector, althoughthe level might have been high or favorable in comparison withthat produced in the native host. Such proteins, which include the 0-lactamase and hemolysin ofB. cereus (214,215,216) streptodornase (211), the xylosidase ofB. pumilis (217), and an amylase of B. stearotheermophilus (203), suggest poor performance of some gram-positive expression signals inB. subtilis. High-level expression of some proteins may be harmful, excluding the use of high-copy-number vectors, but such proteins may be produced with low-copy-number vectors (213,218). There may also be a needto coexpress a gene- and a species-specific regulator gene (205).

7 FUTUREPROSPECTS Due to the successful development of secretion and expression vectors, the productionof heterologous proteins inBacillus for practical purposes is now feasible. Bacillus, epecially B. subtilis, has become a realisticand readily available alternative asa production host for a number of proteins; for some applications,Bacillus can be expected to be the optimal host. The use of bacillar secretion vectors isat its best in production of proteins of nonindustrial strainsand species ofBacillus and of other bacterial proteinsof a secretoryor periplasmic nature.A broad range ofthis type of protein is expected to be secreted effectivelyand thus produced efficiently. As several examples abovedemonstrate, the construction of a

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production systemfor such proteins may be simple and straightforward, thus providing all ofthe benefits of well-established bacillar production technology. This method can be expectedto replace, to a large extent, useof the species of the origin of the protein for production in fermentation processes, especially if the original species is fastidious inits growth condition and difficult to handle, as is nearly always the case for pathogenic microorganisms. The use of effective secretion and expression vector systems is also likely to be far superior in improving the efficiency of industrial production processes compared with the classical methods for improvement of the production organism.It should be notedthat secretion vectors and the technology used for improving their efficiency can also be used to enhance secretionof the homologous secretory proteinsproduced at present in bulk in industry. Although exact figures are not available in the published literature, the present production technology is believed to rely on genetically engineered expression systems in mosttheof processes. Efficient productionof eukaryotic proteins has remained the major challenge for development ofthe expression of heterologous proteinsBacilin lus. However, some eukaryotic proteins have been produced in significant amounts with the present secretion vector systems.It is interesting and encouraging for the future prospects ofthe technology, that these proteins are of pharmacological significance, and in one of these cases, theBacillus production systemwas found to be optimal among those explored. These successful applications, together withother experience, confirmthe possibility of obtaining high yields aofprotein poorly expressed initiallyby the meticulous fine tuningof the appropriate gene constructions,and by determining the optimal growth conditions. The principal obstacleto the secretory productionof eukarytic proteins is their apparent incompatibility with the bacillar secretory machinery. To improve their productionand to extend this technologyto a broader range of eukaryotic proteins it is necessary to introduce more compatibility for the secretion of various kindsof proteins inBacillus. This is a task made quite formidable by the lack of knowledge concerning the molecular biology of protein secretionBacillus in and its unique features, especiallyincomparison to eukaryoticmechanisms.However,many improvements are expected in this regard dueto concerted efforts of research into the mechanism of bacillar protein secretion in several laboratories, together with rapidly progressing studies on the genetics and physiology of this organism.Major components of the secretory machinery

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of B. subtilis have already been identified and characterized, with considerable understandingof the possible or probable bottlenecksof the secretion of heterologous proteins. The identification and cloning of the appropriate gene homolog inBacillus and in the organismof the source of the protein of interest have not only provided tools to identify the source of the incompatibilitybut also suggest suitable means for expression systems of improved compatibility by the addition or replacement ofappropriate components. This progress is also expected to benefit efforts to improve the efficiency of the secretion of those heterologous proteinsalready producible withthe present systems for secretory expression. The way to go is indicated, for example, by the enhanced secretion of exoproteins of B. subtilis achieved by increasing levels ofthe bacillar PrsA protein, therecentlyidentifiednovelcomponentoftheirsecretionmachinery (45), and the inceased level of export of periplasmic proteins in E. coli caused by the hyperexpression ofthe translocation complex proteins SecY and SecE (219). The availability of effective expression vectors has also created conditions for the use ofBacillus hosts for the production of heterologousproteins intracellularly, a possibility so far much neglected in favor ofother production organisms. Experience with a number of model proteins amply demonstrates that Bacillus can served as a host for a very high level of intracellular protein production. The repertoire of present expression vectors is wideand is expectedto provide a modeof production suitablefor any particular protein of interest, with the choice of different temporal Bacillus modes of expression and inducibility. Intracellular production in would share many ofthe features of similar heterologous production systems, but adding the obvious advantages of welldeveloped industrial fermentation technology. The low-level or nonexistence of toxicity and lack of biological activity of bacillar cellular components are further benefits, adding an element of safetyto the fermentation process, contributingto a less demanding purification process,and making proteins produced in Bacillus especially suitable in basic research for studies of their effects and interactions in eukaryotic cellular systems. The frequent formation of inclusion bodies, and thus the nonnative conformation of the protein producedat high levels, isan inherent problem in any intracellular production system. Several proteins have, however, been successfully renatured with methods feasible for practical production, although there are no generally applicable procedures available. The rapid advanceof knowledge ofthe propertiesand structure of manyproteins of interest is also expectedto further such pursuits.

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ACKNOWLEDGMENT I thank Ilkka Palva for critical reading of the manuscript.

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in heat-shock genesgroES and groEL on protein export in Escherichia coli. EMBO J 1989; 8~3517-3521. 19. Lecker S, Lill R, Ziegelhoffer T, Georgopoulos C , Bassford J, Philip J, Kumamoto CA,Wickner W. Three pure chaperone proteins ofEscherichia coliSecB, trigger factor and GroEL-form soluble complexes with prescursor proteins in vitro. EMBO J 1989; 8:2703-2709. 20. Wild J, Altman E, Yura T, Gross CA. DnaK and DnaJ heat shock proteins participate inprotein export inEscherichia coli. Genes Dev1 m 6 1165-1 172. 21. Luirink J, High S, Wood H, Giner A, Tollervey D, Dobberstein B. Signalsequence recognition by an Escherichia coliribonucleoprotein complex. Nature (London)1992; 359:741-743. 22. Miller JD, Bernstein HD, Walter P. Interaction of E. coli Ffh/4.5S ribonu-

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160. Takagi H. Yamagata H, Udaka S. Gene expressionin Bacillus brevisunder control of theLac repressor-operator systemfrom Escherichia coli. J Ferment Bioeng 1992; 74:l-6. 161. Tamura H, Tameishi K, Yamagata H, Udaka S, Kobayashi T, Tomita M, Ikezawa H. Mass production of sphingomyelinase of Bacillus cereus by a protein-hyperproducing strain, Bacillus brevis 47, and its purification. J Biochem 1992; 112:488-491. 162. Edelman A, Joliff G, Klier A, Rapoport G. A system for the inducible secretion of proteins from Bacillus subtilisduring logarithmic growth. FEMS Microbiol Lett 1988; 52:117-120. 163. Nagarajan V, Borchert TV. Levansucrase:A tool to study protein secretion in Bacillus subtilis. Res Microbiol 1991; 142:787-792. 164. Shimotsu H, Henner DJ. Modulation of Bacillm subtilislevansucrase gene expression by sucrose and regulation of the steady-state mRNA level by sacU and sacQ genes. J Bacteriol 1986; 168:380-388. 165. Crutz A M , Steinmetz M, AymerichS, Richter R, Le Coq D. Induction of levansucrase inBacillus subtilis: An antitermination mechanism negatively controlled by the phosphotransferasesystem. J Bacteriol 1990, 172:10431050. 166. Palva I. Production of medically important proteins. Ann Clin Res 1986; 18:337-343. 167. Zukowski MM, Miller L. Hyperproductionof an intracellular heterologous protein in a sacUh mutant of Bacillus subtilis. Gene 1986; 46947-255. 168. Zukowski M, Mille L, Gogswell P, Chen K. Inducible expression system based on sucrose metabolism genes of Bacillus subtilis. Genet Biotechnol Bacilli 1988; 217. 169. Peschke U, Beuck V, Bujard H, Gentz R, Le Grice S. Efficient utilization of Escherichia coli transcriptional signals in Bacillus subtilis. J Mol Biol 1985; 186547-555. 170. Yansura DG, Henner DJ. Development of an inducible promoter for controlled gene expressionin Bacillus subtilis. In: Ganesan AT, HochJA, eds. Genetics and Biotechnology of Bacilli. New York: Academic Press, 1984: 241. 171. Yansura D G , Henner DJ. Use of the Ekcherichia coli lac repressor andoperator tocontrol gene expression in Bacillus subtilis. Proc Natl Acad Sci USA 1984; 81:439-443. 172. Le Grice SFJ, Beuck V, Mous J. Expression of biologically activehuman T-cell lymphotropic virus type I11 reverse transcriptase in Bacillus subtilis. Gene 1987; 55:95-103. 173. Wang LF, Hum WT, Kalyan N K , Lee SG, Hung PP, Doi RH. Synthesis and refolding of human tissue-type plasminogen activator in Bacillus subtilis. Gene 1989; 84:127-133. 174. Leonhardt H, Alonso JC. Construction of a shuttle vector for inducible expression in ficherichia coli and Bacillus subtilis. J Gen Microb 1988; 134:605-609.

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175. Dhaese P, Hussey C, Van Montagy M. Thermo-inducible gene expression in Bacillus subtilis using transcriptional regulatory elements from temperate phage 4105. Gene 1984; 32:181-194. 176. Osburne MS, Craig RJ, Rothstein DM. Thermoinducible transcription system for Bacillus subtilisthat utilizes control elements from temperate phage 4105. J Bacteriol 1985; 163:llOl-1108. 177. Breitling R, Sorokin AV, Behnke D. Temperature-inducible gene expression in Bacillussubtilis mediated bythe I857-encoded repressor of bacteriophage X. Gene 1990; 93:35-40. 178. Diderichsen B. A genetic system for stabilizationof cloned genes in Bacillus subtilis. In: Ganesan AT, Hoch JA, eds. Bacillus Molecular Genetics and Biotechnology Applications. New York: Academic Press, 198635-46. 179. Ferrari FA, Nguyen A, Lang D, Hoch JA. Construction andproperties of an integrable plasmidfor Bucillussubtilis. J Bacterioll983; 154:1513-1515. 180. Fahnestock SR, Saunders CW, Guyer MS, Lofdahl S, Cuss B, Uhlen M, Lindberg M. Expression of the staphylococcal protein A gene in Bacillus subtilis by integration of the intact gene into the B. subtilis chromosome. J Bacteriol 1986; 165:lOll-1014. 181. Janniere L,Niaudet B, Ehrlich P, Ehrlich SD. Stable gene amplification in the chromosome of Bacillus subtilis. Gene 1985; 40:47-55. 182. Declerck N, Joyet P, Le Coq D, Heslot H. Integration, amplification and expression of theBacillus licheniformisa-amylase gene inBacillus subtilis chromosome. J Biotechnol 1988; 8:23-38. 183. Prozorov A, PoluektovaE, Sachenko G, Nezametdinova V, Khasanov F. Various means of integration of the expressible human dihydrofolate reductase gene into the Bacillus subtilis genome. Gene 1987; 57:221-227. Bacillus subtilis: The establishment of 184. Young M. Gene amplification in multiple tandemly-repeated copies of a heterologous DNA segment in the bacterial chromosome. In: Ganesan AT, Hoch JA, eds. Genetics and Biotechnology of Bacilli. New York: Academic Press, 1984:89-102. 185. Kallio P, Palva A, Palva I. Enhancement of a-amylase productionby integration and amplifying the a-amylase gene of Bacillus amyloliquefaciens in the genome of Bacillus subtilis. Appl Microbiol Biotechnol 1987;27: 64-71. 186. Miller J R , Kovacevic S, Veal LE. Secretion and processingof staphylococcal nucleus by Bacillus subtilis. J Bacteriol 1987; 169:3508-3514. 187. Niaudet B, Janniere L, Ehrlich SD. Integrationof linear, heterologous DNA molecules into the Bacillus subtilis chromosome: Mechanismand use ininduction of predictable arrangements. J Bacteriol 1985; 163:lll-120. 188. Shimotsu H, Henner D. Construction of a singlecopy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis. Gene 1986; 43:85-94. 189. Errington J. Gene cloning techniques. In: Harwood CR, Cutting SM, eds. Molecular Biolow Methods for Bacillus. London: Wilev. 1 9 9 0 :

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190. Marston FAO, Hartley DL. Solubilizationof protein aggregates. Methods Enzymol 1990, 182:264-276. 191. Hartley DL, Kane JF. Properties of inclusion bodies from recombinant Escherichia coli. Biochem SOCTrans 1988; 16:lOl-102. 192. Locht C, Keith JM. Petussis toxin gene: Nucleotide sequence and genetic organization. Science 1986; 232:1258-1264. 193. Himanen J-P, Taira S, Sarvas M, Saris P, Runeberg-NymanK.Expression of pertussis toxin subunitS4 as an intracytoplasmic protein inBacillus subtilis. Vaccine 1990; 8:600-604. 194. Sarvas M, KontinenVP, Himanen J-P, Saris P, Taira S, Runeberg-Nyman K. Secretion and production of foreign proteins in Bacillus. In: Heslot H, Davies J, Bobichon L, Durand G, Penasse L, eds. Proceedings of the International Symposium on Geneticsof Industrial Microorganisms 1990 (GIM90), Societe Francaise de Microbiologie, 1990. 195. Nurminen M, Butcher S, Idhpaan-Heikkila I, Wahlstrom E, Muttilainen S, Runeberg-Nyman K, Sarvas M, Makela HP. The class 1 outer membrane protein of Neisseria meningitidisproduced in Bacillus subtilis can give rise to protective immunity. Mol Microbiol 1992; 6:2499-2506. Williams DC, van Frank RM, Muth WL, Burnett JP. Cytoplasmic inclu1%. sion bodies in Escherichia coli producing biosynthetic human insulin protein. Science 1982; 21k687-689. 197. Puohiniemi R, Butscher S, Tarkka E, Sarvas M. High level production of Escherichia coli outer membraneproteins OmpA and OmpFintracellularly in Bacillus subtilis. FEMS Microbiol Lett 1991; 83:29-34. 198. Sarvas M, LehtovaaraP, Sibakov M, KZirihen L. Secretion ofEscherichia coli P-lactamase from Bacillus subtilis by the aid of a-amylase signal sequence. Proc Natl Acad Sci USA 1982; 795582-5586. 199. Hager PW, Rabinowitz JC. Translational specificity in Bacillus subtilis. In: Dubnau DA, ed. The Molecular Biology of theBacilli. New York: Academic Press, 1985:l-29. 200. Wuenscher MD, Klijler S, Bubert A, Gerike U, Goebel W. The gene iap of Listeria monocytogenesis essential for cell viability, and its gene product, p60, has bacteriolytic activity. J Bacteriol 1993; 175:3491-3501. 201. Ortlepp SA, Ollington JF, McConnell DJ. Molecular cloning in Bacillus subtilis of a Baciltus licheniformisgene encodinga thermostable a-amylase. Gene 1983; 23:267-276. 202. Vasantha N, Thompson LD, Rhodes C, Banner C, Nagle J, Filpula D. Genes for alkaline protease andneutral protease fromBacillus amyloliquefaciens contain a large open reading frame betweenthe regions coding for signal sequence and mature protein. J Bacteriol 1984; 159:811-819. 203. Diderichsen B, Poulsen GB, Jdrgensen PL. Cloning and expression of an amylase gene from Bacillus stearothermophilus. Res Microbiol 1991; 142: 793-796.

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204. 205. 206. 207.

208. 209. 210. 211.

212. 213. 214. 215. 216.

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van den Burg B, EnequistHG, van der HaarME, Eijsink VGH, Stulp BK, Venema G . A highly thermostable neutral protease from Bacillus subtilis and characterization of the gene product. JBacteriol 1991; 173:4107-4115. Kubo M, Imanaka T. Cloning and nucleotide sequence ofthe highly thermostable neutral protease gene from Bacillus stearothermophilus. J Gen Microbiol 1988; 134:1883-1892. Fairweather N, KennedyS, Foster TJ, Kehoe M, Dougan G. Expressionof a cloned Staphylococcus aureus a-hemolysin determinant in Bacillus subtilis and Staphylococcus aureus. Infect Immun 1983; 41:1112-1117. Fujii M, Takagi M, Imanaka T, Aiba S. Molecular cloning of a thermostable neutral protease gene from Bacillus stearothermophilus in a vector plasmid and its expressionin Bacillus steurothermophilusand Bacillus subtilis. Bacteriol 1983; 154:831-837. Diderichsen B, Christiansen L. Cloning of a maltogenic a-amylase from Bacillus stearothermophilus. FEMS Microbiol Lett 1988; 5653-60. Jdrgensen PL, Hansen CK. Multiple endo-P-l,4-glucanase-encodinggenes from Bacillus lautus PL236 and characterization of the celB gene. Gene 1990; 93:55-60. Kang JH, Hwang Y,Yo0 OJ. Expression of penicillin G acylase genefrom Bacillus megateriumATCC 14945 in Escherichia coli and Bacillus subtilis. J. Biotechnol 1991; 179-108. Wolinowska R, Ceglowski P, Kok J, Venema G. Isolation, sequence and expression in Escherichia coli,B. subtilis and Lactococcus lactisof the DNase (streptodornase)-encodinggene from Streptococcus equisimilisH46A. Gene 1991; 106~115-119. Kovacevic S, Veal LE, Hsiung HM, Miller JR. Secretion of staphylococcal nuclease by Bacillus subtilis. J Bacteriol 1985; 162521-528. Wang P-Z, NovickRP. Nucleotide sequence and expression of the B-lactamase gene from Staphylococcus aureus plasmid p1258 in Escherichia coli, Bacilltls subtilis and Staphylococcus aureus.J Bacterioll987; 169: 1763-1766. Wang W, MCzes PSF, Yang YQ, Blacher RW, Lampen JO. Cloning and sequencing of the 0-lactamase I gene of Bacillus cereusS/B and its expression in Bacillus subtilis. J Bacteriol 1985; 163:487-492. Sloma A, GrossM. Molecular cloning and nucleotide sequenceof the type I P-lactamase gene from Bacillus cereus.Nucleic Acids Res 1983; 11:4997. Kreft J, Berger H, HWlein M, Muller B, Weidinger G , Goesel W. Cloning and expression in Escherichia coli and Bacillus subtilis of the hemolysin (cereolysin) determinant from Bacillus cereus. J Bacteriol 1983; 159681689. Panbangred W, Fukusaki E, Epifanio EC,Shinmyo A, Okada H. Expression of a xylanase gene Bacilluspumilus of in Escherichia coli and Bacillus subtilis. Appl Microbiol Biotechnol 1985; 22:259-264. Shivakumar AG, Vanags RI, Wilcox DR, Katz L, Vary PS, Fox JL. Gene dosage effecton theexpression ofthe &endotoxin genesof Bacillus thurin-

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Sawas giensis subsp. kurstaki in Bacillus subtilis and Bacillus megaterium. Gene 1989; 79:21-31. Perez-Perez J, Marguez G, Barber0 J-L, Gutierrez J. Increasing the efficiency of protein exportin Escherichia coli. Bio/Technology 1994; 12: 178180. Lundstrom K. Expression of the vesicular stomatitis virus membrane glycoprotein gene in Bacillus subtilis. FEMS Microbiol Lett 1984; 23:65-70. Lundstrom K, Palva I, KaLiiiinen L, Garoff H, Sarvas M. Pettersson RF. Secretion ofSemliki Forest virus membrane glycoproteinEl from Bacillus subtilis. Virus Res 1985; 1985:69-83. Novikov AA, Borukhov SI, Strongin AYa. Bacillus amyloliquefaciens aamylase signal sequence fusedin frame with human proinsulinis properly processed by Bacillus subtilis cells. Biochem Biophys Res Commun 1990; 169:297-301. Vasantha N, Filovla D. Expression of bovine pancreatic ribonuclease A coded by a synthetic gene in Bacillus subtilis. Gene 1989; 7653-60.

3 Gene Expression in Recombinant Yeast Albert Hinnen,” Frank Buxton, Bhabotosh Chaudhuri, Jutta Heim, Thomas Hottiger, Bernd Meyhack, and Gabriele Pohlig CIBA-GEIGY AG, Basel, Switzerland

1 INTRODUCTION A large number of molecular and genetic toolsand techniques have been developed over the past decade which allow the production of a variety of heterologous proteins in the yeast Saccharomyces cerevisiae. Many techniques have been-developed to deliver foreign genetic information to the yeast genome usinga variety of vectors. It is possible to manipulate specific regulatoryand signaling sequencesto ensure propergene expression andto deliver the protein productto the desired location. Thereare essentially no limitations to manipulating this organism at the genetic level. In addition, numerous possibilities existto influence the metabolic flux with the aim of obtaining better production yields. Almost anynew insight into the genetics, biochemistry, and cell biology of this organism has an immediate impact on the work of the yeast genetic engineer. In particular, recent advances in our understanding of yeast cell biology have provided us with a wealth of information and options that can be integrated into the day-to-day approach of the “expression specialist.” ~

*Present uffiliution: Hans-Kndll-Institut fur Naturstoff-Forschung, Jena, Germany.

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Of course, all of this has only been possible as a result of a detailed understanding of yeast geneticsand biochemistry, which was pioneered by a few leadinglaboratories in the 1950s and 1960s, demonstrating the potential of S. cerevisiae as an ideal microorganism for biological studies. S. cerevisiae cells are nonpathogenic, grow rapidly, and have stable haploid and diploid states, which makes genetic analysis straightforward. This, combined with a versatile systemfor unrestricted manipulationof the genome, meansthat S. cerevisiae combines all the virtuesof biological model organism with the long-proven record of a commercial production system. Designing an experimental pathway toward the successful expression of a heterologous gene requires basic knowledge in a number of fields. Even this knowledge is sometimes not sufficient. Predictabilityof success is often frustratingly poor and pragmatic approaches are needed. This means exploring alternativesand combining rational strategies with “gut feeling.” We hope that this chapterwill assist inthe search for new solutions and that it will open upways to explore novel ideas in this still rapidly moving field. We have divided this contribution into two parts. In the first part, we review briefly the basic background in the area of heterologous protein production. Inthe second part we provide data from our own laboratory to illustrate specific points with pertinent examples. Previous reviews have dealt with this subject and related topics. One has been published by Tuite (l), and a valuable monograph dealing with various aspects of yeast genetic engineering has been edited by Barr et al.(2). More basicinformation in Enzymoon yeast geneticsand molecular biology is given Methods in logy, Vol. 194 (3), in the two volumes of The Molecular Biology of the Yeast Saccharomyces (4), and in The Yeasts, Vol. 3 (5). 2

GENERAL

BACKGROUND

2.1 Yeast Vectors Almost all yeast vectorsare artificial hybrids between sequences derived from Escherichia coliand S. cerevisiae. The E. coli part serves as a cloning and amplification deviceand consists of a replication originand a selection marker for this organism. Convenient cloning sites add to the versatility of these vectors (e.g., pUC series). The yeast part varies. A minimal requirement isthe presence of a marker,which allowsfor selection in yeast and a sequence homologousto the yeast nuclear genome. Dependingon

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the additional yeast elements present,the hybrid vectors exhibit specific characteristics with respectto mode of replication, copy number, and genetic stability inS. cerevisiae cells. The general strategyto follow isto find a vector that guarantees a maximal gene dosage without sacrificing genetic stability. Since heterologous gene expression usually represents an additional burden on the cell, the optimal solution is often a compromise.

2. l . 1 IntegrativeVectors Integrative vectorsdo not contain a functional replication origin for yeast (Fig. 1, pFBY4). To be maintained in a growing population they need to integrate into a replicatingstructure (i.e., a chromosome or a yeast plasmid). Integration occurs exclusively by homologous recombination (6). Two types of eventcan.be distinguished: circular donorDNA creates tandem duplications of the target sequence, and linearized DNA leads to an exact replacement of chromosomal or plasmid sequences, provided that both DNA ends are homologous to sequences at the integration site(7,8). As a consequence, integrationcan be directed to any DNA locus byintroducing a double strand break into the donor DNA, resulting in gene replacement (9). Transformation by integration guarantees the highestlevel of stability. Delivery of extra DNA to a yeast chromosome by gene replacement should make such a foreignDNA sequence indistinguishable from a host sequence provided that the host cell is not discriminated against by a significant growth disadvantage. Since circular DNA donorwill create tandem duplications,this will giye riseto looping-out reactions,, low-frequency eventsthat seem to correlate approximately with the length of the duplication (0.1 to 1070 loss inan overnight culture). The copy numbers resulting fromintegration events are 1 for gene replacements andfrom 1to a few for the integration of circular plasmids. Due to the low copy numberattained with integrativetransformation, this mode of gene transfer is not the preferred onefor the expression of heterologous genes. However,there are situations where gene doses have to be low because the foreign gene expression is deleteriousto the host cell, or because overexpression leads to other metabolic effects which reduce the overall efficiency of expression. In thesesituations a single copy integraton can bethe best compromise between gene dosage and genetic stability. An interesting approach involves the creation of multiple insertion even& is applicable only by the use of yeasttransposons (10). Again, this strategy if the foreign gene isnot deleterious to the host cell. Compared withthe 2-pm vector (see below), these multiple integrants should exhibit g higher degree of stability.

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c EcoRl

I 2000

c Sac1 c EcoRI

ECOW

P a t 1 c SphI

Figure 1 (a)

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Figure 1 Yeast vectors for gene cloning and heterologous expression. pFBY4 is an integration vector with the yeast URA3 gene as a selection marker. pDP34 is a 2-pm vector with 2-pm specific functions(REPI, REP2, REP3, FLP, IRl, I=), and URA3 and dLEU2as selection markers for yeast. pGH5 is a centromere vector with CENI4 and ARSl as centromere and replication originsand URA3 and ZLV2-SMR as selection markers for yeast. The vectors above contain pUC18- or pUC19derived E. coli plasmids with the amplicilline resistane(AMP) gene. ILV2SMR is a sulfometuronmethyl-resistant allele of the ILK2 gene.

2.1.2 2-pm Vectors These vectorsare the preferred vehicles for achieving a high level of foreign protein production (1 1). The 2pm vectors are true shuttle vectors that can replicate autonomouslyin E. coli and S. cerevisiae cells (for a detailed overview, see Ref. 12). In addition to the integrative vectors described previously, they containa replication origin derived from the endogenous yeast 2-pm plasmid (Fig. 1, pDP34). This plasmid, with contour a length

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of 2 pm, is a resident of most yeast cellsand its biology has been studied in great detail(13). It is stably maintained without givingan obvious advanDNA”). tage to the host,and it does not have a selectable marker (“selfish The main structural features of 2-pm circlesare the replication origin (OM), a cis-acting stability locus(STB or REP3), and four trans-acting loci (REPZ, REP2, FLP, R A F ) , which are important in plasmid replication/amplification and segregation at cell division (13). Two inverted repeat structures allow switching from one isoform to the othervia a FLP-mediated recombination event (A-form, B-form). Most 2-pm vectors are composed of an E. coli plasmid part, the 2.2-kb EcoRI fragment of the B-form, which REP3 locus, and one of the codes for the replication origin, the cis-acting two inverted repeats. These vectors need a wild-type 2-pm plasmid inthe trans. Withsame host cell(cir’ cells), which provides the other functions in out these functions plasmid stability is poor the andaverage copy number is low. Alternatively, all 2-pm functions (with the possible exception of RAF) can be provided on one plasmid which is then stably propagated without resident 2-pm plasmids (cir’ cells). Stability of the endogenous 2-pm plasmid is very high and segregational loss can not be detected under most growth conditions.All hybrid 2-pm vectors,on the other hand, have markedly reduced stability(1 to 10% plasmid loss after overnight growth). Even higher instabilitycan be observed, depending on the additional genetic load of the vector. The copy number of wild-type 2-pm plasmid isabout 60 per haploid cell, hybrid plasmids have copy numbers ranging from a few (lessthan 5 on average)to more than 50. Obviously, hybrid 2-pm vectors have a large range of copy numbers even under selective conditions. It seems that yeast cells exhibit a high degree of flexibility to adapt to the best tolerated copy number,which is the resultof a positive selection pressure for plasmid retention via the complementing geneand a negative selection pressure for plasmid loss exerted by expressing the foreign gene (“metabolic burden”)(14). For foreign geneproducts that donot affect cell growth, a number of genetic trickscan be appliedto boost copy number. The best known system makes use of a defective allele ofLEU2 which acts as a complementing marker in aLEU- host (11). This LEU2 gene (leu2-d allele) is devoid of its own promoter and is transcribed from a read-through transcript orifor growth, ginating in>pm sequences. To provide sufficient gene product the gene dosage (i.e.,the plasmid copy number) has to be increased(15). The net resultof this selection pressure is the preferential growth of cells with increased copy number. A similar system has been by devised Unternahrer et al. (16) on the basis of a temperature-sensitive CDC9 allele.

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Yeast cells witha deleted CDC9 gene are transformed with a 2-pm vector containing the temperature-sensitive cdc9-l allele (17). Under low-temperature conditions the copy numbernotisaffected since even a low gene dosage of cdc9-l is sufficientto support growth. When the temperature is shifted to above 30°C,only cells with higher copy numbers can survive. This selection pressure leads to a higher average copy number the in growing population because a higher copy number results in more active CDC9 gene product(DNAligase), which inturn can compensatefor the destabilized protein. Surprisingly enough, these high copy numbers are maintained over many generations even in the absence of selective pressure, probably becausethere are sufficient trans-actingfactors available to support a high level of replication and amplification. An additional advantage of this system isthat the plasmid cannot be lost from the cell because yeast cells cannot grow without DNA ligase. As indicated above, all these vectors contain E. coli plasmid sequences which are needed for cloning and amplification in E. coli but do not serve 3 we address this point and any function in the final yeast host. In Section describe an interesting strategy to remove these dispensible sequences via a homologous recombination event in vivo. The resulting vectors have better stability than that of conventional hybrid vectors.

2.1.3 OtherVectors There are a number ofadditional yeast vectors available, but their role in biotechnological applications is relatively minor. The most prominent ones include centromere vectors (Fig. 1, pGH5) and artificial chromosome vectors. In both cases a functional yeast centromere is included, which leads to a copy number of approximately1 (18,19).They are most useful for the isolation and functional analysisof yeast genes (centromere vectors) (20) and mammalian DNA sequences (artificial chromosome vectors) (21). 2.1.4 SelectionSystems Most vectors utilize genes involved thein biosynthesis of amino acids and nucleotides. Usually, the vectors carry the wild-type genesand the hosts have the corresponding auxothrophic mutation. Stable mutations (deletions, double and triple mutations) are available for all commonly used loci. The obvious disadvantage of these vectorsthe is need for the introduction of auxothrophic mutations into the strains of interest. A few dominant resistance genesare in use, but most scientists preferthe amino acid and nucleotide complementing genes since selection can be made abso-

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lutely tight, whereas spontaneous resistant clones usually arise with dominant resistance markers.The most commonly used markers are summarized in Table1. Vectors carryingURA3 or L YS2 are very useful because yeast mutations inthe URA3 or LYS2 gene can easily be obtained by selecting for resistance to 5-fluoroorotic acid(41) or a-aminoadipate (42), respectively.

2.2 Transcription Efficient and faithful transcription can only be obtained from yeast transcriptional control sequences. These include promoter sequencestranand scription termination signals, most conveniently arrangeda transcripin tion cassette. If additional control sequences are placed within the cassette (e.g., protein coding regions as signalsfor protein targeting), the more general term expression cassette is used. Genetranscription depends on the 5’ and 3’ sequences flanking the protein coding region. Since relatively little is known about factors that stabilize/destabilize the mRNA transcript (43) and since the foreign DNA coding sequence is in most cases fixed, our discussion will concentrate primarily on transcriptional elements that determine transcription initiation. For the sake of their utility for heterologous protein expression,one can distinguish between constitutive promoters and regulated promoters.

2.2.1 Promoter Elements and Transcription Terminator Elements Many ofthe elements of yeast promoters are typical for eukaryotic genes (Fig. 2; for a review, see Ref.44).Starting from the protein-initiating ATG (position + 1to + 3) and moving upstreaminto the promoter region, these elements include the mRNA start site (-30 to -60), a mRNA selection site (about 5 to 15 bp upstream fromthe start site), a TATA box(-60 to -160) and one or more upstream activation sequence (VAS) elements (about 60 to 100bp upstream fromthe TATA box). UAS elements are cis-acting elements that mediate transcription activation. Theyare analogous to the enhancer sequences found in higher eukaryotes. The spacing between these elements and their sequence content appear to be less stringent in yeast than in higher eukaryotic promoters. This allowsfor a larger flexibility in fusing the foreign DNA sequence to the yeast control sequences. For convenience most fusions are done between the corresponding mRNA start sites and the beginning ofthe protein coding regions. This is accomplished by providinga yeast promoterarm, which ends with a restriction site closeto the initiating ATG.

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Table 1 Genetic Markers Used for Yeast Transformation

Refs. Yeast genes complementing auxotrophic mutations LEU2

HIS3 TRPl u . 3

Vector (examples)

pYeleulO pJDB219 Yep13 pDP34' YEpL3 pYehis3 YEP6 pYc 1 YRp7 PLC544

-

Pa2 pDP34' YEP24 pFBY4' L YS2 ADE2 Yeast genes conferring resistance phenotype CAN1

CUP1 TUNR

IL V2-SRM CHY2 E. coli genes conferring resistance phenotype cat hYg kan %e Fig. 1.

pYRp3R pASZlO TLC-l =p36 pJR41 pGHS'

22, 6 11 34 23 24

25 26 27 28 29 30 31 23 26 This laboratory 32 33

pRCl

34 35 36 This laboratory 37

p m 11 pLG89 pRC2

38 39 40

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,+-- AAA

H

60

L. .I

60-160

AUG UCU ---+mRNA

b

Figure 2 Structural elementsof an efficient yeast promoter and the corresponding ATG context region.UA'S, upstream activating sequence;TATA, typical eukaryotic transcription-initiation controlregion;CnTn,pyrimidine-richtrack; ATG, initiation codon.

Transcription termination is recommended to ensure contained transcription. Although many reports exist that demonstrate efficient expression without added transcription termination signals, it is not advisable to rely on it. 2.2.2 ConstitutiveExpression

The most straightforward approach to express a heterologous gene in yeast is to take a strong promoter, which is efficiently expressed in the presence of glucose. These include promoters controlling the genes coding for the glycolytic enzymes 3-phosphoglycerate kinase (PGK) (49, gly(GAP/TDH3)(W,enolase (ENO) ceraldehyde-3-phosphate dehydrogenase (47), triose-phosphate isomerase( T ' Z ) (48), and the fermentative enzyme alcohol dehydrogenaseI (ADHZ)(49). These genesare often called constitutive; however, one has to keep in mindthat expression is repressed to various levels when cells are grownon nonfermentable carbon sources. For many applications constitutive expression is the best solution, since the fermentation process is easier to handle and expression titers are largely growth related. Combined with high-copy-number 2-pm vectors, expression levels of several percent total of soluble protein can be obtained. We will not cover these promoters in any detail since extensive coverage of the most prominent representatives (PGK, GAP/TDH3) is available (50,51).

2.2.3 RegulatedExpression Regulated expression is necessary for products that interfere with cell growth. It is usually not possibleto determine the point of interference,

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and various terms have been used to describe this phenomenon of growth inhibition (toxicity, metabolic burden, metabolicload). Expression from a regulated promoter allows uncoupling of growth from production and thus may delay synthesis untillater in the growth phase and/or reduces the time periodof production to a few hours. Since most of the available regulated promotersare less efficientthan the constitutive promoters described above, expression levelsare usually lower. Furthermore, the fermentation process may becomerather complicated and the system may be fully exploitable only with sophisticated feeding regimesand control devices. Shake flask experiments will often not reveal the full potentialof these expression systems. The most commonly used regulated promoters are galactokinase/galactose epimerase(GALI, GALZO) (52), acid phosphatase (PH05)(53,54), the regulated alcohol dehydrogenase gene (ADH2) (S), and the CUP1 system (56) (see also Section 4). In addition, hybrid promoters are available that combine promoter elements from strong conconstitutive promoters(downstream elements) with UAS elements from regulated promoters (upstream elements) (23,55,57). a. The GAL System. This systemis designed to metabolize galactose and has been studied extensively (58). Several structural genes (GAL2, GALI, G A L 3 coding for a permease and various conversion enzymes are needed to direct galactoseinto the glycolytic pathway. The GAL system is regulated twofold:it is repressed by glucose and induced by galactose. The best studied genes are GALI and GALZO, organized in a diverA number of regulatory loci control gene gently transcribed gene cluster. expression. The GALA protein binds at a UAS region between GAL1 and GALZO and, in the presenceof galactose and absence of glucose, induces transcription. In the absence of the inducer galactose, GAL80, a negative.factor, bindsto the activation domain ofGALA and thus abolishes transcription. The GAL system is tightly regulated and can be induced up to 1000-fold (48,59). Under high-copy-number conditions there seems to be a limitation inthe positive regulatoryfactor GALA, which can be GAL4 at the same time. Since overexpresovercome by overexpression of sion of GALA increases the background expression under repressed conditions, yeast strains have been constructed withan integrated GAL4 gene regulated by the GALZO promoter (60). b. The p H 0 System. Phosphatases in S. cerevisiae are organized ina gene family consisting of individual members which are regulated bythe is PH05, presence or absence of inorganic phosphate. The best studied gene coding for a secreted acid phosphatase(61,62). In termsof transcriptional

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control the system exhibits obvious similarities to the GAL system described above. Two positive factors, PH02 and PH04, are needed to activate transcription.PH04 is negatively affected byPH080, which may interact directly with PH04 to abolish transcription in the presence of high levels of inorganic phosphate. Additional regulatory genes include PH08.5 and PHO81. Regulation by phosphate is an interesting option since it allows transcriptional control irrespective of the carbon source (23,54,62). As with the GAL system,there is a need to control the depletion of the repressor (glucose or phosphate, respectively). This is best achieved under controlled fermentation conditions.An alternative is the use of a yeaststrain with a temperature-sensitive activator, pho4’”. At the restrictive temperature 37”C, the pho4’ gene product is inactive, and at lower temperature, 23”C, induction can take place. In the presence of a pho80 mutation, induction occurs irrespectiveof the presenceof inorganic phosphate in the medium by a temperature downshift(63). c. The CUPI System. S. cerevisiae contains a single metallothionein protein that is encoded bythe CUPl gene (64). This locus is responsible for conferring copper resistance on yeast cells by a combinatio of CUPI gene amplificationand CUP1 transcriptional induction following the addition of exogenous copper. A UAS promoter region has been defined where the positive factor ACEl (CUP2) binds.ACEl itself binds copper in a highly cooperative fashion. The CUPl system is the only natural yeast system in whichtranscription is activated by the addition of a small molecule, in this case Cu2+, tothe medium. It is somewhat surprising that this system has been used only rarely for the production of heterologous proteins (56). In Section 4we provide an example of hirudin expression under control of the CUPI promoter.

2.3 Translation of Heterologous mRNA Inspection of the 5’ untranslated leader region of yeast genes reveals that this sequence is nonrandom. There is a strong bias for A residuesand almost no G residues are found. It is probable that this fact is important for the expression of heterologous proteins since the 5‘ sequences seem to influence expression levels of foreign proteins dramatically without affecting the steady-state levels of mRNA (65). This is in agreement with (CYCI),where modificationof the 5’ data from homologous yeast genes leader sequences (addition of G residues) leads to inhibition of translation (66).

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In higher eukaryotic genes the ATG context region is very important in controlling the efficiency of translation initiation (67). This is also true 5’ AAAfor yeast, but the consensus sequence is substantially different: AUG-UCU 3’ (68). In particular, the -3position seems to be very important. As a whole, however, the need to conform to the consensus is less stringent in yeast compared with higher eukaryotic cells. S. cerevisiae genes showa codon bias(69) which is most prominent for highly expressed genes.It is speculatedthat this has consequencesfor the expression of foreign genes, since codon bias seems to parallel the abundance of tRNA isoacceptors. Data in the literature are very scarceand do not allow firm conclusions to be drawn. Only in a very few cases have careful comparative studies pointed to an effect of codon exchanges(70). There is, however, general agreement that rare codons (usuallyG/C rich) should be avoided(CGA, CGG, CGC, GGA, GGG, GCG, CCG, CCC, GUA, CUC), although yeast hasthe capability to translate all 61 amino acid-specific triplets (71). For a review on yeast protein synthesis, see Ref. 72. 2.4 Secretion of HeterologousProteins

One major attraction in choosinga fungus for heterologous protein production isthe potential of these microorganisms to secrete large amounts of protein productsinto the medium.Protein secretion uses the synthetic capacity of the cell and at the same time removes its product from the synthetic machinery. This is ofparticular importance for toxic products that interfere with structural or enzymatic components in undesirable ways, which isprobably the caseat high expression rates. Since commercial applications tend toward ever-increasing titer demands, these considerations almost always apply. As a eukaryotic organism,S. cerevisiae shares allthe major characteristics of the mammalian secretory pathway (73). It is therefore likely that proteins of mammalian originwill have a good chance of being properly secreted from yeast cells. There are, however, specific differences between lower and higher eukaryotes aswell as within these twobroad classes of organisms. The most important, from a practical point of view, is the rather different capacityfor posttranslational modifications (see below). 2.4.1 Secretion Signals Proteins destinedfor secretion have N-terminal extensions the in form of signal sequences (presequences), which range from approximately 15 to 30 amino acids, microbial signals usually being at the lower end of this

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size range. These signal sequences may be followed by prosequences which have large size variations. Additional sequences may be present as C-terminal extensions. Commonto all these sequences is their transient nature. While the role of praequences is that of a primary signal for passage through a membrane, prosequences may have a multitude of functions, ranging from auxiliary signal sequences to folding or antifolding moieties and enzymatic inhibitors. In eukaryoticcells membrane passage takes place at the level of the endoplasmic reticulum, where initial processing steps and protein folding occurs. The folded protein is then transported through a series of membrane compartments until it is finally released to the exterior of the cell. Due to the close similarity of the secretion pathways of eukaryotic cells it is not evident whether a secretion signal should stem from the expression host or from the foreign gene product. Literature data show that both strategies may be followed. If a foreign proteinis secreted inits homologous host it will probably encode a signal sequence. In the case aof simple presequence this N-terminal extensionwill probably fulfillthe same role in a nonhomologous environment. It is our experience that taking the signal sequenceof the foreign protein is a good starting point. Alternatively, signal sequences derived from yeast proteins may be used. Frequently used yeast presequences are the ones derived from the yeast invertase (SUC2, 19 amino acids), acid phosphatase(PH05,17amino acids), and the yeast a-factor pheromone (MFeZ, 20 amino acids). In yeast, the most commonly employed N-terminal extension with signaling function is the a-factor leader of the e-mating factor precursor. It comprises a 20 amino acid presequence followed by a 61-amino acid prosegment. Thea-factor leader serves, together with the spacer elements, to direct secretion and processing of four units of 13-amino acid mating pheromone peptide (Fig. 3). Despite the frequent of the usea-factor leader sequence, there is no reason to give this signal preference overshort presequences, except for the secretion of smaller peptides (see below). The requirementfor all of the above pre-and prosequences is twofold: they haveto act as secretion signals and they have to be removed efficiently and accurately after doing their job. Unfortunately, predictability for these requirementsis low, although some empirical rules seem to emerge. For size considerationsseems it logicalthat small peptides (e.g., hormones) need additional peptide spacers in order to emerge from the ribosomes before terminationof their synthesis. Since membrane passage occurs cotranslationally, additional amino acids are needed to ensure couplingof synthesis with translocation into the endoplasmic reticulum. Furthermore,

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pre- and prosequence cleavages are enzymatic processes with target sequence as well as steric requirements. Signal sequence removaland precursor processing involvea complicated cascade of enzymatic reactions, starting with the actionof the signal peptidase located in the endoplasmic reticulum followed by endoand exoproteolytic cleavagesat a late Golgi or secretoryvesicle compartment. Of critical importance is endopeptidase F (product of the KEX2 gene) which cuts at the C-terminal sideof a dibasic amino acid motif. Although these enzymatic steps are found in all eukaryotic organisms, subtle differences at the boundaries between homologous and heterologous sequences may explain incomplete or inaccurate processing or signal sequence removal. The first reported caseof foreign protein secretion from yeast cells was the production of human interferon (74). Since then many proteins and peptides have been secreted using a variety of signaland leader sequences(23,47,75-77), some of them already reaching commercial exploitation (78,79). The field of heterologous protein secretion from yeastiscells very complex and reflects the difficulties encounterednot only in synthesizing a protein but also intransporting it via a complex network of membrane structures involving several enzymatic processing steps. More basic knowledge in cell biology and membrane traffic is needed before more rational approaches can be designed. In Section 5 we discuss the production of the leech peptide hirudin by secretion from yeast cells. 2.4.2 PosttranslationalModifications Apart from signal sequence cleavage and other proteolytic maturation steps, additional modifications that are linked to secretion are important. The more prominent ones are the covalent additions ofN- and O-linked sugars (for a review on glycosylation in yeast, see Refs. 80 and 81). Very little is known about the addition of sugars to the hydroxyl groups of serine and threonine; moreinformation is availableabout the process of N-glycosylation. N-Glycosylation in S. cerevisiae follows the same rules as in higher eukaryotic cells(82). A core sugar moiety is transferredto the Namide of asparagine in the sequence context Asn-X-Ser/Thr, with X being As in higher eukaryotic cells this core sugar any amino acid except proline. undergoes additional modification steps in the endoplasmic reticulum and in the Golgi apparatus, the major difference in S. cerevisiae being the absence of the trimming enzymesand modification enzymes adding complex sugars at the Golgi level, which in higher eukaryotes results in the formation of shortened side branches ending with complex sugars (galactose, sialic acid, fucose).S. cerevisiae cells instead'maintain the core

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mannose scaffold, often extended with a large amountof additional mannose residues, which can lead to a substantial increase in the molecular weight ofa protein. Variousmutants are available that block specific glycosylation steps inS. cerevisiae. The most interestingare mnn9, which is responsible for outer-chain additions, and mnnl, which encodes a termnnl minal al,3-mannosyltransferase.Double mutants having defects in and mnn9 have therefore been suggested for use (83).However, sincespecific glycosylation patterns are important features of mammalian proteins and, in addition, wrong sugars may represent immunogenic determinants, S. cerevisiae cells are less suited for the productionof mammalian proteins with complex sugar structures. This is particularlyso with proteins of pharmaceutical interest.

2.4.3 Secretion and Protein Folding The folding of secretory proteins is posttranslational a event taking place in the endoplasmic reticulum, where the environment is particularly favorable for disulfide bridgeformation and the generationof higher-order structures. It is therefore understandable that the secretion mode may represent the only solution to generate a correctly folded foreign protein. This view has been substantiatedby several examples(23,83,84). A number of proteins havebeen identified as being involved in this process,and most of them have also been described for S. cerevisiae. They include protein disulfide isomerase(PDI) (85),the KAR2 gene product (86),prolyl isomerase (87),and immunophilins (88).Very little is known about their role in catalyzingthe folding stepsof foreign proteins. Since proper protein foldingseems to be a prerequisitefor efficient secretion(89), the process of attaining the correct secondary and tertiary structure is crucial. The questionof whether foreign proteins need their homologous counterpart to facilitate an efficient folding process remains to be answered. The role of chaperonins (90) as facilitators or inhibitors of structure formation will most certainly receive moreattention in the field of heterologous protein production. In this context it is worth considering that the prosequence of thea-factor precursor couldwell function as a “folding agent” a neutral extension ofa secretion signal. for foreign proteins and not as just Production of properly folded proteinsis one of the important areas of application ofS. cerevisiae as an expression host.It has obvious technical advantages over mammalian systems and it still outweighs other microbial eukaryotes, due to the extensive knowledge ofthe genetics and cell biology of the system. Since E. coli cells do not readily secreteproteins across their inner and outer membranes that organism does not pre-

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sent a strong alternative.All these reasons makeit attractive to study the S. cerevisiae intensively withthe aim to exploit protein folding process in this feature further. An experimental approach to study the folding path7. way of the human insulin-like growth factor1 in yeast is given in Section

2.5 Stability of Heterologous Proteins Stability of heterologous protein products has received relatively little attention in the past. The reason for this is probably an overestimationof factors that control the early steps of the expression pathway (replication, transcription, translation). This inturn may reflect the fact that parameters controlling these steps are easier to understand and to approach at the molecular and genetic levels. It is, however, quite clear that in many cases failure to obtain adequate levels of heterologous proteins is due to rapid protein turnover. InS. cerevisiae two different proteolytic degradation systems are known. One is the vacuolar degradation system,which contains a numberof unspecific proteases(91); the otheris a nonvacuolar system with a variety of specific peptidases, probably coupled to the ubiquitin system (92).

2.5.7 GeneralPrecautions Many production hosts carry amutation in the gene coding for proteinase yscA, often in the form of the pep43 mutant allele, which, however, exhibits a leaky phenotype.Protease yscA is located in the vacuole and is the major processing protease responsiblefor the maturation of other vacuolar proteasesand other hydrolases (92,93). A defect inyscA therefore affects other potentially harmful proteolytic enzymes,in particular the vacuolar proteinaseyscB. The role of vacuolar proteinases in general protein turnover is still poorly understood but probably involves an as yet unknown import process of proteins destined for degradation (94).

2.5.2 ProteolyticDegradationintheCytoplasm The main degradative system in the cytoplasm seemsto be theATP- and ubiquitin-dependent pathway generally found in all eukaryotes (95). It involves the conjugation of ubiquitin (50 to 76 amino acid peptides)to Part of the degradative machinery proteins that are subsequently degraded. is probably the proteasome particle, a multifunctional enzyme complex which contains among other activities protease yscE (96). The initial sigpathnal that channels a protein to the ubiquitin-dependent degradation way is not yet understood. Interestingly enough, it has been shownthat protein degradation is a function of the amino-terminal residue (N-end rule) (97) and follows the same ubiquitin-dependent pathway(98).

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2.5.3 Protein Degradation in the Secretory Pathway There isno firm evidencefor a general protein degradation system the in secretion pathway. However, thereare a number of well-defined proteolytic activities either residing the in secretory membrane systemor which are transported through this system. Inaddition, all known vacuolar hydrolases travel viathe endoplasmic reticulumand parts of the Golgi apparatus to their final destination. Inaddition to yscA and yscB, two carboxypeptidases, yscY and yscS, are fairly well characterized. Carboxypeptidase yscY is synthesizedas a precursor that is activated in the vacuole only by proteolytic maturation steps, including yscAand yscB (99,100). The enzyme exhibits a broad-specificity spectrum and has been studied extensively (91). The gene codingfor yscS has been cloned and sequenced (101). Surprisingly, yscS does not need activation by yscA or yscB, and thus seems to travel through the secretory pathway asan active enzyme. Other proteases inthe secretory pathway include the two processing pep(91). They are encoded by the genes KEXZ and KEx2, tidases ysca and yscF respectively. These membrane-associated proteases are involved in the ,maturationof the a-factor precursor andof the killer factor (Fig. 3). yscF

STE13

KEXI

Figure 3 The or-factor precursor consists of fouror-factor repeatunitsand hasan Nterminally extended pre- and prosequence. Processing occurs in the endoplasmic reticulum (signal peptidase cleavage) and at a late Golgi or secretory vesicle state. KEX2, endoproteolytic digestion after the dibasic cleavage motif Lys k g ; STE13, dipeptidyl aminopeptidase cleavageto digest N-terminal spacer amino acids; KEX1, carboxypeptidase digestion to remove the C-terminal amino acids Lys and k g .

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is a protease located in thelate Golgi apparatus or the secretory vesicles (102). It cleaves at the C-terminal side ofa pair of basic amino acid residues and as such is a necessarypart of the processing systemfor protein fusions, including the a-factor leader sequence. ysca is a carboxypeptidase witha high specificity for basic amino acids (103). Like yscF, it is membrane associated and localized to later stages of the secretory pathway. Its biological role is the removal of the dibasic aminoafter acids cleavage by yscF. Mutants of KEX1 and KEX2 are perfectly viablebut exhibit a sterile phenotype. Other proteinaseswhich are fully secreted and therefore potential candidatesfor proteolytic degradationof secreted proteins are theBAR1 gene product (104), a pepsin-like proteinasethat inactivates a-factor peptide and aminopeptidases yscll (91). While many of these proteinases serve specific functions and as such exhibit a narrow specificity range, they can become crucial in situations where large amounts of foreign proteinsare being produced (see Section 5). In addition, it is certainly possiblethat high-level production ofproteins may represent a stress signalfor the cell which leadsto the inducttion of a variety of proteolytic activities (92).

3 STABLE YEAST 2-pM VECTORS 3.1

Introduction

As mentioned in Section2.1.2, the native 2-pm plasmid, being stableand is ideal vectorfor present at approximately 60 copies per haploid cell, an the expression of high levels of foreign protein. Thusit is not only surprising but also disappointingthat most engineered 2-pm vectors display such poor stability (Ref. 105 and our unpublished observations). However, mostof these vectors were, with the benefit of hindsight, very badly constructed, having deletions interruptions or in oneor more of the functional regionsof the 2-pm sequence. In recent years, engineered full-length 2-pm vectors having all essential functions intact have been constructed. We and others (106,107) have found that bacterial sequences present on 2-pm-derived plasmidsare bad for plasmid stability, as can be seen by comparing plasmidspFBY 12Rand pFBY 12PR with PFBY8R (Table 2). Plasmid pFBY8R does not contain bacterial sequences when propagated in yeast, whereasthe other plasmids do. Whether the instability is caused by sequences acting as yeast promoters (108-110) or for some other sequence-specific effect isnot clear. It is not simply becausethe inclusion of bacterial sequences makes theplasmids'larger, since plasmids of the same size but lacking bacterialDNA appear to be more stable.

Two-bm-Derived Plasmid St FRT

Plasmid

1

2

pFBY12 pFBY12P pFBY 12R pFBY12PR

-

+

pFBY8R pFBY99 pF~Y96

+

-

+ + + +

-

SnaBI cCDC9 URA3 < CDC9 < URA3

-=

+

U.3> CDC9> U M 3 > CDC9>

+ + +

CDC9> U M 3 >

-

Insert at: BgIII

-

I

-

-

_.


3.u

>

-

ght/left 1.17 3.28 1.17 3.28 2.31 1 .02 1.8

Plasmid-free cells (070) 17 43 4.0 6.5 0.9 1.3 3.2

aThe plasmids listed were all derivatives of A (Fig. 4). Some have a small deletion in either FRTI or FRT2, indicated by a minus sign. Each plasmid also carries a number of genes inserted into either the SnaBI, BgIII, or BamHJ sites. The order of the genes at each site is shown clockwise starting at FRT2 and the direction of transcription of each gene is shown: > is clockwise and < is anticlockwiseas the plasmids are drawn in Fig. 4. GHIR symbolizes an expression cassette for hirudin driven by a truncated GAPDH promoter. Note that those plasmids that have three intact FRT sites will recombine as soon as they are transformed into yeast, thus excising and rapidly losing the bacterial sequences. The ratio of the distance between the Xbal sites in the intact FRTsites present in yeast is indicated. Note that the right segment is that which contains the origin of replication. The percentage of plasmid containing yeast cells after 24 h of growth in rich media was determined by plating aliquots on rich media followed by replica plating onto defined media.

5

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Our results and thoseof others ( 1 11,112) have also shown that the presence of promoter activity affects2-pm plasmid stability.Part of the difference in stability between pFBYSR and either pFBY99 or pFBY% can be accounted for by the strong truncated GAPDH promoter presenton the latter two plasmids (Table2). How transcription mediatesthis reduction of stability is not clear, butit appears to be inpart caused by protein binding at the promoter regions ( 1 12). Avoidance of strong plasmid-borne promoters is, however, not favored if these vectorsare to be used for protein expressionin an industrial context. Even with this increased understanding of the engineered 2-pm plasmids, it is still apparent that these vectors are not as stable as the native 2-pm plasmid and, perhaps more important, are often not stable enoughto be used in large-scale batch or continuous-culture fermentation. Given that the 2-pm plasmid itself is so stable, we felt that it should be possible to improve the stability of engineered 2-pm plasmids.

3.2 Designing Plasmids and Effects of Symmetry on Plasmid Stability Our plasmidscontain all of the 2-pm sequencesso they can be cultured in a cir" host. To put inserts into these plasmids that do not interrupt the known functional regions, others have successfully used the SnaBI site next to the ORI(106,113)or the HpaI site inthe STBlocus (107).We have used the SnaBI in preference to the HpaI site as the latter separates the distal and proximal halves ofthe STB locus.This is not per se deleterious, as STB proximal can stabilize the plasmid independentlyof STB distal unless transcription initiated within the insert runs through STB proximal (114). Other possible sitesfor insertion are between the FLP and REP2 genes or between the RAF and REP1 genes. However, we have not explored these regions betweenthe divergent transcripts, as theyare quite short (1 15) and must containthe binding regionsfor the regulatory complex formed betweenREP1 and REP2, with the possible involvement of RAF ( 1 16,117). Thus a priori one might expectthat inserts in these regions would be deleterious, and its has been shown that inserts into the XmaIII site between the FLP and REP2 genes lead to plasmid instability (107, 113). The other sitesthat we have used were engineeredinto the plasmid to give convenient cloning sites, BamHI and BgIII in Fig. 4, at the 3' ends of the messages of FLPI and REPI. Both of these sites are actually in the 599 bp inverted FRT repeats ( 1 H ) , but they werenot expected to interfere withthe FLP-mediated recombinations,as it is knownthat only

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A

Recombination between FRTl and FRTP in yeast

/

J

Rapidly lost from yeast

Figure 4 A is a physical map of a plasmid that forms the basis of most of the plasmids described in this section. It consists of all of the 2-pm DNA (-) integrated into pTZ18R (=) in such a way as to duplicate the F'T sites (FRT1 DNA. Reand FRT2) as direct repeats separating the bacterial- and yeast-derived

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about 20 bp in the center ofthis region are required for in vitro function (110). We have shownthat inserts in these sites do not affect FRT-mediated recombination or plasmid stability. This The plasmidsthat we have studied also lack all bacterial sequences. can be achieved by cutting and ligating the E. coli-propagated plasmids before transforming them into yeast to remove the bacterial sequences (120), or more elegantly, by flanking the bacterial sequences with FRT direct repeats so that on transformation into yeast, these recombine to yield two plasmids, one containing a single FRTcopy and all the bacterial sequences and the other a 2-pm derived plasmid that is free of bacterial sequences (Fig. 4) (106,121-124). We have used the latter technique,having duplicated a 166-bp FRT-containing site on both sides of the bacterial vector sequences (Fig. 4). The double-rolling-circle model of2-pm plasmid copy-number amplification first proposed by Futcher (125) suggeststhat the ORI should be as close to one of the FRTs as possible, and the other FRT sites should be as distant from thisas possible, to maximize the probabilityof the initiating recombination event occurring after the first FRTsite is replicated and before the other site is replicated. This organization of the FRTs and ORI are conserved in other 2-pm-related plasmids which are found in a variety of other yeasts(126-132) showing almostno sequence homology to one another. It is clear that in all the engineered 2-pm plasmids, this symmetrical positionof the FRT sites in the plasmid is disrupted since all the foreign DNA sequences are integrated into a unique site inthe plasmid. We have shownthat maintaining this symmetrical disposition of the FRT sites in engineered plasmids leads to improved stability, as shown in Table 2. (Compare pFBY12 and pFBYlZP, which differ by 20-bp de-

combination between these two sites, mediated by FLP, general recombination pathways, or in vitro, leads to the plasmids B and C. The latterwill not be maintained in yeast,as it does not contain a yeast origin of replication, consisting merely of the bacterial vector pTZ18Rand a 166-bp fragment containingan FRTsite from 2-pm yeast. However, B will be maintained in yeast in two forms, one as shown and one that is a simple inversion, by recombination between the indirectly repeated FRT sites. The long arrows indicateORFs in this plasmid. The little arrows indicate the orientationof the FRT repeats, and OR1 indicatesthe yeast origin of replication. This basic plasmid has been modified in numerous ways by insertions of different pieces of DNA into some or all of the sites markedor by deletions to make other, more useful plasmids, some of which discussed in this section.

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letions around the XbaI sites presentin the FRT loci and also pFBY 12R and pFBY 12PR.) The more symmetricalpartner of each pair the is more stable. Similarly, when propagated in yeast, pFBY99 and pFBY96 differ only by the position of a 136-bp FRT-containing fragment located either before or after the URA3 gene. In this pair the more symmetrical derivative is also the more stable.

3.3 Use of Stable Plasmids for Heterologous Protein Production We have used these plasmidsas vectors for the expression of both hirudin and insulin-like growth factor 1. In the former case the stable symmetrical plasmids from which bacterial sequences had been deleted resulted ina yield increase of 100to 140% compared with a host-vector system based on plasmid, pDP34. These results from shake flask cultures reflect the good baseline stability of pDP34 carryinga hirudin expression cassette. However, pDP34-derived plasmids carrying IGFl expression cassettes are dramatically unstable with greater than 30% loss inan overnight culture in rich media. Plasmids constructed so as not to contain bacterial sequences and which are also symmetrical show less than a 1.5% loss overnight and over a 200% increase in IGFl titer. 3.4 Conclusion

The 2-pm-derived plasmids are more stable if they: 1. Contain the complete 2-pm sequence and are culture in ciro cells. 2. Have allthe genes and other important regions, FRT, ORI, promoters, and so on, intact and use the SnaBI, BgIII, or BamHI sites at the 3' end of the shown in Fig.4. (One could also engineer a site REP2 gene without deleterious effects,but we have not tried this.) 3. Have no bacterial sequences. (This is achieved most easily by exploiting the FLP recombinase.) 4. Are symmetrical. (We stilldo not know how symmetricalthe plasmids haveto be, butless than 20% difference in the two armsis probably desirable.) 5. Have no inexplicable destabilizing sequences. (The difference in stability between pFBY12and pFBY12R, which differ only by the orientation of the inserted DNA, is large. This instability ofpFBY 12 can be abolished by the deletion of just 4 bp at the end of the insert next to the 5' end of the URA3 gene. There appears to be no consistent explanation for the data concerning this region.)

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It is still apparent that although we have plasmids which are stable enough 1 0 7 0loss inabout 10generafor practical industrial use, exhibiting less than tions on rich media, the rate of loss is still much larger than that seen for native 2-pm plasmid at approximatelyper cellpergeneration.This implies that there is still a lotto be learned aboutthe stability of the 2-pmbased plasmids.

4 CUP1-DIRECTEDHETEROLOGOUSEXPRESSION IN YEAST 4.1

Introduction

The usefulness of yeastas a host for the production of recombinant proteins depends largely on the availability of efficient and versatile promoter systems. In Table 3 we summarize the characteristics of some of the more widely used yeast expression systems (for more detailed information, see Ref. 55 and Section 2.2). According to Table 3, one of the best promoters for use inan expression vector is that of the yeast metallothionein gene, CUPZ. The promoter strength is very high (and can even be adjusted to the requirements of a particular process),the system is genetically well defined, and its application is technically feasible in processes involving large fermentersand complex media. Nevertheless,the CUPZ promoter has so far been used almost exclusively in academic research. In this sectionwe introduce the CUPZ-system and describe its successful application in the production of recombinant hirudin in yeast (see also Section 5). Several comprehensive reviewson metallothioneinsas well as a number of papers dealing withthe construction of vectors for the CUPZ-directed (56,64,133-135). expression of heterologous proteins have been published Therefore, only the essential features of the CUPZ-system are discussed below. Metallothioneins are small, cysteine-rich metal-binding polypeptides widely distributed among eukaryotes(64). S. cerevisiae contains a single metallothionein proteinthat is encoded bythe CUPZ gene (136-138). The CUPZ locus has been shown to confer copper resistanceto yeast cells(139141). Two natural variants of S. cerevisiae with respect to copper resistance have been described: Strains sensitive to 0.3 mMcopper contain a single copy ofthe CUPZ locus andare designated cupZs;strains resistant to 0.3 mM copper are designated CUPP and contain several tandemly repeated copies of the CUPZ locus (136). Copper resistance relies on a combination ofCUPZ amlification and CUPZ translational induction

ie 3 Characteristics of Yeast Expression Systems

Promoter strength ( S ) and tightness

C)

ADH 11

S : One of the strongest promoters currently available. Directs the synthesis of ca. 5% of total protein/mRNA in a homologous situation T: 30-fold induction upon shift from acetatelpyruvate to glucose. Basal level of expression too high to allow the production of toxic proteins. Shift from acetate/ pyruvate to glucose feed.

S: Directs the synthesis of ca. 1% of the soluble protein in a homologous situation. T: mRNA undetectable in the repressed state.

Both S and T are excellent.

Glucose depletion.

Involves release from glucose repression and induction by galactose.

I

Induction mech~ism

GALI, GAL7, CALI0

PGK

PHU5 S: Superior to GAP S: Lower than that of strong constitutive promoter (this promoters (e.g., GAP). study). Can be increased by T: mRNA barely dethe construction of tectable in the rehybrid promoters. pressed state. 30- to Promoter regulation 50-fold induction by is partially lost in hycopper addition. brid promoters. Promoter tightness T: mRNA undetectable regulatable by changunder repressed condiing the number of tions. Ca. 40-fold inCUP1 copies in cells. duction by shift from + P to - P medium. Phosphate depletion. Addition of a copper salt to the medium.

Feasibility/physiological consequences of promoter induction

Manipulations required for promoter activation are simple. Nowever, the cells have to be pregrown on, e.g., acetate or pyruvate. On these carbon sources, growth is relatively slow.

No exact control over time point of inducduction. Promoter induction will also induce proteases.

Promoter regulation

Complex, incompletely understood.

Adjustability of expression levels

No.

Complels (involving catabolite repression), but well defined. No.

Use of complex media

OK.

OK.

No exact control over the time point of induction if cells are grown on glucose. This problem may be overcome by introducing a mutation that results in release from catabolite repression. Galactose is an expensive inducer. See ADHII.

No exact control over anipulations for the time point of inpromoter induction duction. ~ ~ o s p h a ~ e are simple, can occur depletion i n c o ~ p a t ~ ~ at ~ eany time, and have with growth. Increased relatively minor efprotease activity under fects on the host. induced conditions.

Complex, but well defined.

Relatively simple (only two genes involved).

Possible, but difficult since the inducer is also a substrate for growth. OK.

No.

Possible.

Variable phosphate content of media components (e.g., peptone) may create problems.

OK.

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following the addition of exogenous copper (136-138). A &-acting Sequence (UASJ necessary to promote copper-inducible transcription of the CUPZ gene has been identifiedand was mapped to positions -105 to -148 upstream of the transcriptional start site of CUPZ (142-144). Footprint analysis and electrophoretic mobilityshift assays revealed the binding ofa cellular factor to UAS, (143,144). Further analyses showedthat the binding factor is the product of the ACEZ (= CUP2) locus, which is essential for the copper-induced transcription of the CUPZ gene (143145). The ACE1 protein is a transcriptional activator that binds copper (and to a lesser extent, silver) ions in a highly cooperative fashion (143, 146). Metal bindingalters its conformation, thereby activating its DNAbinding domain. The conformational change of ACElp eventually allows the CUPZ gene to be transcribed. If it is to be used as a vehicle for the expression of heterologousproteins, an important feature of the CUPZ-system is its autoregulation. A yeast strain in which the CUPZ locus was deletedfrom the chromosome showed asubstantial level of transcription from a plasmid-borne CUPZ: galKreporter gene even ifno copperwas added to the medium (147). Copper control of CUPZ transcription in a ACUPZ strain was restored by reintroducing a functional CUPZ gene under thecontrol of a constitutive promoter (148). A detailed study by Wright et al. (149) has shown that this autoregulatory behaviorof yeast metallothionein dependson its ability to bind metals. Thus CUPlp appears to repress its own synthesis by complexing free copper ions in the cell, which, in turn, interferes with ACElp activation. This is important for biotechnological applications in that changing the numberof chromosomal copiesof the CUPZ gene may result in altered levels of cloned gene expression (148).

4.2 Use of the CUPI-System in the Expression of Recombinant Hirudin Variant HV1 in Yeast We made use ofthe CUPZ promoter to direct the synthesis and secretion 5 and Ref. 14) in yeast. of recombinant hirudin (variant HV1, see Section The plasmids constructedfor this purpose are shown in Fig.5. They are based on plasmid pDP34, which contains the full 2-pm complement. The pH05 signal sequence fused in frame to expression cassette consists of the the hirudin coding region and a PHO5-derivedterminator (14), all placed under the control of either atruncated version of the strong, constitutive GAP promoter (plasmid pDP34/GAPFL-YHIR) or the CUPZ promoter (Plasmids pPFY56, pPFY58,and pPFY59R). Plasmids pPFY58and

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pPN59R contain, in addition to the hirudin cassette, the ACE1 gene, either under itsown promoter (pPFY58)or fused to the CUPl promoter an appropriate host strain, (pPN59R). The plasmids were transformed into and the transformants were cultivated essentially as described in Section 5. Except for precultures, complex medium was used in all experiments. In a first step, the appropriate time point for the induction of the CUPZ expression cassette was determined in shake flasks. A fixed concentration of copper sulfate (200 CClM) was added to the cultures either (a) immediately after inoculation, (b) during the early exponential phase,(c) late in the exponential phase, (d)at the onset of the diauxic lag-phase, or(e) during the early stationary phase. Irrespective of the type of plasmid used (pPFY56, pPFY58,or pPFY59R), the highest titers of hirudin (as determined by HPLC analysis) were measured in the cultures supplied with copper rightat the time of inoculation.In cells transformed with plasmid pDP34/GAPFL-YHIR, the addition of copper to the medium didnot significantly alter hirudin titers compared with controls without copper. Next, the optimal copper concentrationfor hirudin expressionwas determined. Two different host strains were used, which contained either 3 (strain TR1456) or 10 (strain TR1631) copies of the CUPZ locus. As shown in Fig. 6, approximately 1.5 mM copper (supplied at the time of inoculation) was optimal for hirudin production in strain TR1456. The concentration optimum was the same for all types of plasmid used (pPFY-56, pPFY58, or pPFY59R). Instrain TR1631, the optimal copper concentration was shifted to slightly higher values. However, the maximal hirudin yields were significantly lower when TR1631 was used as the host. This is probably due to the autoregulatory behaviorof the CUPI-system as described above (see also Ref. 148). Strain TR1631 is expected to express the CUPl protein at higher levels than the more sensitive strain, TR1456. This will reduce the concentration of copper ions availablefor ACElp activation, thereby causing the CUPZ-directed expression of hirudin to decrease. Hirudin titers obtained withCUPl the expression plasmids were compared to those measured when using plasmid pPD34/GAPFL/YHIR containing a hirudin cassette under the control of the constitutive GAPFL promoter (dashed line inFig. 6). It is evident that the use of the CUPZ promoter considerably improved hirudin yields. This wastrue not only in shake flasks but also in fed batch fermentationup to the 30-L scale. an optimized versionof the fullNote that the GAPFL variant is already length GAP promoter (14). Thus a regulatable promoter may be superior to a strong constitutiveone even if, as in the case of hirudin, the heterologous protein to be expressed is relative nontoxic to the host cell.

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TR1456IpPFY56

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Figure 6 Hirudin productionby strains TR1456 (three copies ofthe CUPZ locus) and TR1631 (10 copies of the CUP1 locus). Both strains were transformed with the hirudin expression plasmids shownin Fig. 5. The cultures were grown on complex mediumfor 72 h, and hirudinwas determined by HPLC in theculture supernatant. Copper sulfate at the indicated concentrationswas supplied at thetime of inoculation.

Unexpectedly, the presence ofthe ACEI gene on the expression plasmid reduced rather than increased hirudin titers. The lowest titers were obtained with plasmid pPFY58, which contains a copy of the ACE1 gene under its own promoter (Fig. 6). The poor performanceof pPFY58 was, at least inpart, due to the fact that it was lostat a much higher frequency than pPFY56 or pPFY59R, albeit only in the presenceof copper in the medium. In contrast, a plasmid related to pPFY58 but with the hirudin cassette underthe control of the GAPFL instead of the CUP1 promoter was stably maintainedeven inthe presence of copper. Thus the deleterious effect of ACE1 on plasmid stability appeared to be mediated bythe CUPIhirudin expression cassette. We tested whether pPFY58 promoted the synthesis of hirudinat such a high rate that the secretion capacity of cells became limiting. However, no clear indication of a secretion blockwas found. The definite reason for the instabilityof pPFY58therefore remains to be established.

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In contrast to pPFY58, plasmid pPFY59R was maintained almost as to pPFY56 in terms stably as pPFY56. Nevertheless, it was slightly inferior of hirudin production. One possible interpretation of this findingis that the presenceof ACE1 on a multicopy plasmid increasesthe intracellular concentration of CUPlp. This would reduce theamount of free copper ions, thereby causing down-modulation of CUPI-directed gene expression. This hypothesis is currently being testedby reducing the numberof CUPl copies in the host.

4.3 Conclusions The CUPZ-system is one of the most attractive yeast expression systems currently available. The CUPl promoter was used in a pseudoconstitutive way to direct the secretion of recombinant hirudin in yeastand was found to be superior to the strong constitutive GAPFL promoter in both shake flasks and fermentors.

5 YEAST PROTEASES INVOLVED IN DEGRADATION OF RECOMBINANT HIRUDIN 5.1

Introduction

Hirudin, originally isolated from the blood-sucking leechHirudo medicinalis, is the most potent inhibitor of the protease thrombin knownto date (150). Sincethrombin playsan important role not only inthe coagulation cascade but also in the activation of platelets, ultimately resulting in fibrin clotformation and platelet aggregation, inhibition of its activity is believed to provide an important tool for the treatment of various thrombotic disorders. Several isoforms of hirudin have been isolated, all of which contain 65 or 66 amino acid residues, six cysteine residues, and have molecular weights of approximately 7 kDa (15 l , 152). The five N-terminal amino acid residuesare hydrophobic, followedby the core region linked through three disulfide bridges. The C-terminal region is rich in acidic residues, which are important for the binding of hirudins to the recognition exositeon thrombin. Inthe natural molecules, the tyrosine at position 63 is postribosomally modifiedto a tyrosine-O-sulfate.

5.2

Secretion of Recombinant Hirudin into the Medium

To produce sufficient quantities of active inhibitor for clinical evaluation, we decided to express one ofthe variants, HVl, in S. cerevis'iae. A syn-

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thetic gene was constructed which contains the yeast acid phosphatase (PH05) signal sequence fused in frame to the coding region of mature W1 in preferred yeast codon usage. The gene was placed underthe control of a truncated 198-bp-long fragment of the yeast GAP promoter (14), and the construction was completed by addition aofPHOS-derived terminator fragment. The entire expression cassette was either inserted into the multicopy 2-pm-based vector pJDB207 (23) for the transformation of appropriate cir+ yeast strains or pDP34 (23), which contains the full Zpm complement for the transformation of ciP yeast strains. Precultures of the transformed yeast strains were grown under selective conditions in minimal medium; the main cultures were generally grown under nonselective conditions in YPD-based complex mediafor up to 96 h. Hirudin expression was followed with a colorimetric assay measuring thrombin inhibition. A typical fermentation.pattern of a strain transformed with a hirudin expression plasmid is shownFig. in 7. Under the conditions chosen, hirudin is produced ina strictly growth-dependent manner,as one could

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Figure 7 Fermentation of a hirudin-producing yeast strain in YPD-based complex medium. Hirudin was analyzed in a thrombin-inhibition assay.

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expect for the constitutive GAP promoter fragment. Also, the vast majority of the hirudin produced is secreted into the medium, and only a minority, most probably in a transient form, is found inside yeast cells. Using the PH05 signal sequence, large amounts of biologically active hirudin can be secreted into the yeast culture medium. Analyzing the culture broth by high-performance liquid chromatography (HPLC), one major and two minor peaks were observed at retention times where hirudin variants should elute (Fig. 8A). The three proteins were isolated by a combination of adsorption on a macroreticular resin, ion-exchange chromatography, and gel filtration. Fast-atom bombardment/mass spectometry (FAB-MS) gave molecular weights of 6963, 6832,and 6719 for peaks 1, 2, and 3, respectively. Comparison with the molecular weight of native W1 and further protein characterization studies showed that S. cerevisiae is not able to carry out the sulfation reaction at tyrosine 63 which leads to the production of desulfato-HV1 in yeast. Full-length desulfato-HV1 (hirudin 65) corresponds to the major peak, peak 1 on HPLC, whereas peak 2 lackthe two minor peaks represent C-terminally truncated forms, ing the C-terminal glutamine (hirudin64) and peak 3 lacking in addition the penultimate leucine (hirudin 63).

ProteolyticDegradation of Desulfato-Hirudin In Vivo C-terminal truncation of proteins expressed at high level ina recombinant host could bethe result of premature transcription terminationor the result of posttranslational proteolytic degradation by host proteases. Lack of one or two amino acidsat the C-terminal end of hirudin could point to the involvement of yeast proteases, resident in the secretory pathway. We therefore crossed a strain lacking proteinase yscB, carboxypeptidase yscY, and carboxypeptidase yscS (bys232-31-42; Table 4) with a strain lacking carboxypeptidase ysca (96; Table 4). Spores were selected, auxotrophic for leucine and each lacking all but one of the three carboxypeptidases. Also, one spore was isolated lacking all three carboxypeptidases. Four strains were transformed with the pJDB207-based hirudin expression plasmidand analyzed for hirudin production byHPLC. For this experiment a semisynthetic medium containing casamino acids was used (153) which had previously been shownto enhance formationof the byproducts. Figure 9A showsthat thestrain with mutations in yscY, yscS, and ysca is completely devoid of the truncated hirudin 64 and hirudin 63 by-products. Figure9B-D represent the pattern obtained with spores 5.3

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A

Figure 8 HPLC chromatogram of secreted hirudinspecies: (A) yeast strainH449 (KEXZ, PRCZ); p)yeast strain TR1456 (prcZ::ura3. kexZ::ura3). Samples were loaded on a C,, reversed-phase column and eluted with an acetonitrile/trifluoroacetic acid gradient.

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Table 4 Yeast Strainse

Name of %

bys 232-31-42

H449

TR1456 MATa, TR1303 MATa, TR14%

Ref.

MATa, adel, kexl, thrl MATa, prbl-l, prcl-l, cpsl-3,

lys2, leu2, his7 MATa,cpsl, prbl, leu2-3,112, ura3A.5, c i f prbl, cpsl, prcI::ura3AS, kexl::ura3A5, leu2-3,112, ura3A.5, c i f cpsl, prbl, kexl::ura3A.5, pral::ura3A.5, leu2-3,112, ura3A.5, c i f M A Ta, prbl, cpsl, kexl::ura3A5, prcl::ura3A5, pral::ura3A5, leu2-3,112, ura3A.5, c i f

166 166

Our collection

Our collection Our collection

Our collection

'"he gene disruptions in PRCl and KEXI were carried out as described in the text. The disruption in thePRAl gene was generated accordingto Pohlig et al. (167).

that all contain one of the three carboxypeptidases. Figure 9B indicates that yscS has no effect underthe experimental conditions chosen, whereas yscY dramatically degrades hirudin (Fig.9C). Interestingly, the presence of ysca, the gene productof KEXZ, also leads to the formation of hirudin 64 and 63 (Fig. 9D), although to a lesser degree. Based on this experiment, C-terminal truncation of hirudin expressed in S. cerevisiae can be assignedto the activityof two yeast proteases, yscY andpm.yscY resides inthe yeast vacuoleand is known to have arather broad substrate specificity (154). ysca! is an integral membrane protein in a late-Golgi compartment, being involved inthe maturation of the precursors of yeast pheromone a-factQrand the killer protein (103). In both natural protein substrates ysca sequentially removes the dibasic lysine-arginine amino acids exposedat the C-terminus after the initial ysc-F-mediated endoproteolytic cleavage step. The crossing experiment indicatesthat in the case of hirudin, ysca obviously also seems to accept leucineand glutamine as substrates. To gain further insight into the unexpected proteolysis of hirudin by the two proteases,we tried to localize the degradation process in the different carboxypeptidase mutant strains. For this experiment the supernatant medium of the untransformed strains fermentedas described

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flgure 9 HPLC chromatograms of secreted hirudin species in carboxypeptidase mutant strains: (A) (prcl, cpsl, kexl); (B) (prcl, kexl, CPSI); (C) (cpsl, kexl, PRCZ); (D) (cpsl, prcl, KEXI).Samples were analyzed by reversed-phase HPLC as described in Fig. 8.

above was incubated with pure hirudin in vitro and the occurrenceof degradation measured by HPLC. Surprisingly, most of the yscY-assigned hirudin-degrading activity wasfound outside the cell, in a non-cell-associated form. Although active yscY is supposedly found only in the vacuole of the cell, our findings support earlier observations by Stevens et al., pointing to a partial mislocalization of yscY to the cell surface, where the precursor can be processed into an active form (155). YSCa-mediated proteolysis was not found outside the cells, which is consistent with the enzyme being an intracellular, integral membrane protein.

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5.4 Hirudin Degradation in a Strain Disrupted in PRC7 and E X 7 To prove the direct involvement ofthe two carboxypeptidasesyscY and ysca! inthe C-terminal proteolytic degradation of hirudin, we sequentially disrupted their structural genes PRCZ and KEXZ, respectively, in the host strain H449 (Table4). A 2.6-kb CIaI/pvUII fragmentof the genomic PRCZ locus from yeast (156) was subcloned into the NarI/SmaI sites of pUC19. The resulting plasmid was cutat the unique StuI site and a blunt-ended 1.2-kb HindIII fragment containing theURA3 gene wasinserted. pUC19/ prcZ::URA3 was digested withAatII and the fragment usedto transform a ura3- yeast strain. Uracil-independent transformants were tested for correct PRCZ disruption by Southern blotting and again made auxotrophic for uracil by 5-fluoroorotic acid-induced selectionfor ura3- segregants. For disruption of KEXZ a 1.4-kb HindII/BamHI fragment containing the 5’ part of the KEXZ gene (157) was isolated from total genomic yeast DNA and subcloned into pUC19. The blunt-ended 1.2-kb HindIII fragment containingthe URA3 gene was inserted into the unique EcoRV site. pUC19/kexZ::URA3 was digested withHindIII/BamHI and the fragmentused to transform the Ura3-, PRCZdisrupted strain as described above. The 5-fluoroorotic acid-induced selection for Ura- segregants of correctly disrupted Kexl- strains was repeated and the resulting Ura3- strain transformed with a pDP34-based hirudin expression plasmid. The €PLC chromatogram of the strain disrupted in PRCZ both and KEXI is shown in Fig.8B underneath its isogenic parentalstrain shown in Fig. 8A. Incontrast to the parental strain, the disruptedstrain no longer produces the two C-terminal degradation by-products. Intermediate strains, lacking either one of the two carboxypeptidases, contain still measurable quantities of hirudin 64 and hirudin 63 (data not shown). The sequential disruption of the two protease encoding genes PRCZ and KEXZ therefore confirms the result obtained in the crossing experiment and provides proof of the direct involvement ofthe two carboxypeptidasesyscY and ysca! in C-terminal hirudin degradation.

5.5 Influence of Endoproteases on Hirudin Production 5.5.7 Proteinase yscA Expression of heterologous proteins in S. cerevisiae is commonly achieved by useof transformed proteinase yscA-deficient strains (e.g., pep4-3 mutants) (91). Strains lacking yscA accumulate inactive precursors of pro-

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teinase yscBand carboxypeptidaseyscY and show reduced activitiesin a number of other vacuolar hydrolases (99,100,158-162), which consequently results in a significant reduction of overall proteolysis. Since hirudin is C-terminally degradedby carboxypeptidaseyscY, we have comparedthe effects of a disruption in the yscA encoding genePRAZ [PRAZ and PEP4 refer to the same gene (161)] with a direct disruption of the yscY structural gene PRCZ. Figure 10 shows the secretion of hirudin 65, hirudin 64, and hirudin 63 by the isogenic strains TR1456, TR1303,and TR14% (see Table 4)dura complex medium basedon YPD up to 168 h.Strain ing fermentation in TR1456 (PRAZ;prd; kscl) as well as TR1303 (PraZ; PRCZ; kexl) produce exclusively hirudin 65. The lackof C-terminal hirudin degradation is due to the absence of active carboxypeptidase ysca as well as yscY, sinceboth the disruption of PRCZ and the disruption of PRAZ lead to lack of active yscY. However, the total amount of hirudin 65 produced bystrain TR1303 is about 30% lowerthan the amount produced by strain TR1456. This result suggests that a strain containing yscA produces more hirudin than does a yscAdeficient counterpart. This assumption is supported by the observation that additional disruptionof PRAZ in TR1456 leadsto a similar reduction in hirudin titers as observedfor TR1303 (Fig.1OC). The reduction in hirudin titer is not to due growth defects of TR1303 and TR14%, since growth of the yeast strains compared isvery similar in the stationary (Figs. 10 and 11) and logarithmic phases (data not shown). It has been reported that strains lacking yscAare not only severely impaired in their ability to undergo sporulation but alsoshow a dramatically increased loss of viability undernutritional stress conditions such as nitrogen starvation (163). This observation suggests that even though the eliminationof proteinase yscA activityand the consequent eliminationof other proteolytic activities is not lethal to the cell,yscA exerts major functions in the stress

64.

Figure 10 Production of hirudin compounds hirudin 65, hirudin and hirudin 63 by yeast strains:(A) TR1303 (prul::ura3,PRCI,kexI::uru3);(B) TR1456 (PRAI,prcl::ura3, kexl::ura3);(C)TR1496 @ral::urajl,prcl::ura3, kexI::ura3) during fermentation in complex medium. Strains were transformed with plasmid pDP34 containing the hirudin expression cassette. Samples were withdrawn from the cultures after various periods of time, the cells removed by centrifugation, and the supernatants subjected to HPLC. In parallel, the optical density of the cultures was measured at600 nm.

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response. Consequently,we conclude that yscA mightnot be dispensable for the optimal performanceof a yeast strain that is usedfor production of heterologous proteinsat high expression levels. Results confirming thisnotion were obtained by fermenting the three isogenic strains in a semisynthetic medium containing casamino acids, which appears to induce an increase in proteolytic processes (153). Again, the strains lacking yscA activity have a significantly lower titer of total hirudin than that of the strain exhibiting yscA (Fig. 11). However,contrast in to the results obtained in complex medium, we can observe C-terminal hirudin degradation inTR1303. This proteolysis seemsto be due to carboxypeptidase yscY, since it cannot be detected in TR1496, in which in addition to PM1 and KEXI, PRCl is disrupted. Thus surprisingly, uncan be detected in a strain lackder certain growth conditions, active yscY ing yscA. There are two possible explanations for this observation: (a) the precursor of yscY is partially activeor (b) the precursor is activated independently of yscA under certain conditions. Stevenset al. reported PEP4-independent processingof yscY in the periplasmic space of strains overproducingyscY (155)or mislocalizingyscY to the cell surface in vplmutants (164). At this stage we do not know whether our finding can be connected withthe processing protease postulated by Stevens et al. However, since allour strainsused carry a mutationin the PRBl gene (see Table 4), we can rule out proteinase yscB as the potential processing agent of yscY under our conditions. 5.5.2 Proteinase yscB Proteinase yscB has been reported to be a major source of protease problems during biochemical studies ofyeast (93,165). Therefore, we have expressed hirudin mostly in yscBdeficient strains(see Table 4). To examine whether yscB really hasan impact on hirudin degradationor, like yscA, can positively influencetotal hirudin production, we generated an isogenic derivative of TR1456, in which the mutated copy of PRBl, the yscB-encoding gene, was replaced byan intact copy ofthe gene. Preliminary results suggest that yscB does not cause degradationof hirudin. On the other hand,

Figure 11 Production of hirudin compounds hirudin 65, hirudin 6 4 , and hirudin 63 by yeast strains: (A) TR1303 (pral::ura3,PRCI,kexl::ura3);(B) TR1456 (PRAI,prcl::ura3, kexl::ura3); (C)TR14% @ral::ura3,prcl::ura3, kexl::ura3) during fermentation in minimal medium. Strains were transformed and fermentation samples withdrawn and treated as described in the legend to Fig. 10.

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Hlnnen et al.

the presence ofboth yscB and yscA does not amplify the positive effect of yscA alone. No further increase in hirudintiter can be observed. We are currently investigating whether the performance of a yeast production strain that lacks yscA can be improved by the presence of yscB.

6 OVERSECRETION IN YEAST 6.1 Introduction Secretion allows the convenient relocation of proteins outside the cell. The efficiency of the secretion processis an important factor for highlevel expression of secreted proteins. In comparison to filamentous fungi and higher eukaryotic cells,the total amount of protein secreted in yeast is rather low, which raises the question of whether secretion in yeastis rate limiting. Indeed, the issueof secretion capacityis controversial (168). It seems necessary to consider secretionof genuine yeast (homologous) proteins separately from secretion of foreign (heterologous) proteins. The homologous situation clearly involves fewer parameters, which could affect the secretion capacity the of yeast celland therefore seems to be easier to analyze. Secreted yeast proteins such as acid phosphatase or invertase are released into the periplasmic space and are mostly retained there for the benefit of the cell. Nevertheless, some periplasmic enzymes reach the extracellular medium, probably as a result of passive leakage during bud formation (169). There is evidencethat anincreased gene dosage for acid phosphatase and invertase from 2-pm-based multicopy vectors leads to higher enzyme activitiesbut does notsaturate the secretory pathway (170, 171). We describe here, however,a systemthat clearly defines a subpopulation of cells with intracellular accumulation of yeast acid phosphatase. This is associated with a nongrowth phenotype.

6.2 Expression of AcidPhosphataseinYeast Overexpression of acid phosphatasewas achieved by replacingthe inducible promoter of the acid phosphatase gene(PH05) (172) by a strong, (GAP49 = TDH3) (173,174) and cloning the constitutive yeast promoter new expression cassetteinto the multicopy yeast vector pDP34 (23). The new expression plasmid is referred to as pDP34/GAPFL-PHOS (Fig. a poorly expressed 12). Plasmid pDP34 has an URA3 selection marker and dL.EU2 gene (leu24 allele) without itsown promoter (15,175). Yeast strain YB18 (MATa, his3-11, his3-15, ura3A5, leu2::ura3ASEY[cir"])was trans-

Figure 12 Expression plasmid pDP34/GAPFL-PHO5. The pH05 expression cassette was inserted between BamHI and Sal1the inmulticloning site of the yeastE. coli shuttle vector pDP34(23) in an anticlockwise orientation. Arrows indicate length and direction of open reading frames. REPli REP5REP3 (= STB), IR1, IR2: 2p elements (12);AMP, p-lactamase gene;URA3, yeast URA3 gene; dLEU2, defective (promoterless) yeastLEU2 gene (14,15); PH05,acid phosphatase gene without promoter region; GAPFL, 201-bp fragment of the promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene(GAP49 = TDH3; see Refs. 14 and 46). Restriction sites in italics were usedfor cloning, and sites in brackets were lost by cloning steps. 165

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formed with the expression plasmid pDP34/GAPFL-PHO5 or with the control plasmid pDP34. Selectionfor the defective leu2-d marker should result ina higher average plasmid copy number to complement adequately the gene dosage, the expresthe feu2 defect of YBl8. Concomitantly with sion of acid phosphatase should increase.

6.3 Cellular Response to Overexpression of Acid Phosphatase Transformants of YB18 (YBl8/pDP34/GAPFL-PHO5) were cultured under uracil selection. Midlog cells were collected and shifted to a medium with either uracil or leucine selection. Cells under uracil selection grew as well as control cells, and the total acid phosphatase activity (Fig. 13), as well as its cellular distribution (Fig. 14), were as expected. Most of the activity was secreted into the periplasmic spaceand to a small extent into the medium.A small fraction of the activity was detected intracellularly. In contrast, under leucine selection, cell growth stopped (despite slow a increase in optical density). The total acid phosphatase activity per cell was comparably high (Fig. 13)and the cellular distribution changed (Fig. 14). The nongrowth phenotype was not caused by the selection for leucine per se, sincecontrol cells grew wellunder leucine selection(Fig. 13). Further analysisby subfractionation showed that under leucine selection acid phosphatase accumulated to a considerable degree intracellularly (ca.60% of radiolabeled, immunoprecipitated acid phosphatase); a smaller part (ca. 35%) was secreted to the periplasmic space and less than 5% Flgure 13 Growth of YB18 transformants under uracil (-ura)or leucine (-leu) selection and total acidphosphataseactivity. YB18 cells transformed with pDP34 (left) or pDP34/GAPFL-PHO5 (right),respectively,weregrown at 30°C to logarithmic phase in minimal medium supplemented with leucine. were Cellsharvested by centrifugation (2000g), washed, and resuspended(0.4 OD,> in minimal medium. The cell suspension was then dividedand supplemented with either leucine ( m a ) or uracil (-leu). The cultures were grown at 25°C for 24 h at 180 rpm. At different time points aliquots were removed and assayed for optical density (ODm) and acid phosphatase (APase) activity according to Haguenauer-Tsapis and Hinnen (170). Acid phosphatase activitywas determined in the culture supernatant, in the periplasmic space (assay with nonpermeabilized cells), and as cellassociated activity [assay with ethanol/toluol permeabilized cells (170)l. Intracellular acid phosphatase activity was the difference between cell-associatedand periplasmic activity.Total activity was the sum of intracellular, periplasmic,and extracellular activity. Enzyme activity is expressedU, inmol/h/ODm.

acidphosphatase periplasmicspace APase activity [Km01 / h / OD6001

7 65-

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time(h) acidphosphatase intracellular 8

APase activity [ p m 0 1 / h / ODsw]

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reached the medium (Fig. 15). With the same techniquethe distribution of acid phosphatase in cells under uracil selection 30% was (intracellular), 55% (periplasm), and 15% (medium) (Fig. 15). Acid phosphatase as 12sites for N-linked glycosylation (89). The degree of glycosylation an is indicator for intracelluar localizationof a protein. Acid phosphatase synthesized under leucine selection was mostly cell associated and highly glycosylated, if not slightly overglycosylated. This indicates that even the intracellularly accumulated fraction of the acid phosphatase had passed the Golgi, probablyat a slow rate of transport, resulting inan overglycosylated protein. Under uracil selection only mature acid phosphatase was found. The expressionlevel of acidphosphatase increases with the gene dosage in the cell (170). We therefore assume, by way ofextrapolation, that the intracellular accumulationof acid phosphatase under leucine selection is a consequence of high gene dosage. Due to unequal distribution of 2-pm plasmids in a cell population(13), it is rather unlikely that all cells show the same effect,but it could be limitedto a subpopulation of cells only. Immunofluorescence analysis of individual cells is a means to address a subpoputhis question. Acid phosphatase indeed accumulated only in lation of cells. A fraction of cells (ca.10%) exhibited a punctuated (sometimes diffuse) staining, suggesting Golgi localization of the intracellular acid phosphatase. I t is a logical conclusionthat such a subpopulation of cells with a high plasmid copy number also is present in the growing cell culture under uracil selection. Intracellular accumulationof acid phosphatase ina subpopulation of cells resulted inthe aberrant, exaggerated cellular structures seen with the electron microscope. These structures resembled -among others -Berkeley bodies (176) and exaggerated endoplasmic reticular structures. Cytochemical staining of acid phosphatase indicated that acid phosphatase accumulated in those structures. Also, the actin pattern in the cells was changedto diffuse surface staining, and the chitin deposition appeared on the cell surface instead of in the bud region. The higher density of cells with intracellular accumulation of acid phosphatase was used to enrich this subpopulation on a LudoxAM density gradient(176). The majority of cells (9oVo) from the bottom fraction of the gradient had acid phosphatase, which had accumulated intracellularly (immunofluorescent label). Cells Figure 14 Distribution of acid phosphatase (APase) activity in ell cultures grown under uracil (-ura) and leucine (-leu) selection conditions. Acid phosphatase activity was determined in the periplasmic space, within the cells, and in the culture supernatant as described in Fig. 13.

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ngUm 15 Radiolabeling and subfractionation of acid phosphatase. yB18/pDP34/ GAPFL-pH05 transformants were grown under uracil (-uracil) and leucine (-1eucine) selection conditions as described in Fig. 13. After 12 h of fermentation the cells (2 OD,) were harvested and radiolabeled essentiallyaccording to Riederer and Hinnen (89) in 400 p1 of minimal medium without uracil (-uracil) or leucine (-leucine), respectively, for 30 min with 100 pCi [%]methionine (NEN: loo0 Ci/ mmol) and chased for 15 min in the presence of 2 mM methionine. The labeled cells wereseparated from the culture medium (m) by centrifugation. Theells were further incubated with lyticasefor 1 h. After centrifugation the supernatant(p = periplasmic fraction) was collected and the pelleted spheroplasts (i = intracellular fraction) were resuspended incitrate buffer.Acid phosphatase was immunoprecipitated from the subfractions m, p, and i with a polyclonal antiacid phosphatase antibody. The resulting immunoprecipitateswere dissolved, treated with 1 mU of endoglycosidaseH (Boehringer, Mannheim)at 37°C overnight, and analyzed by SDS-polyacrylamidegel electrophoresis. The fluorogram is shown. Subfraction m (lanes 1 and4) represents extracellular acid phosphatase,fraction p (lanes 2 and 5) periplasmic enzyme, andfraction i (lanes3 and 6) intracellular acid phosphatase.

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from the bottom fraction were able to synthesize acid phosphatase under uracil and leucine selection, in contrast to cells from thetop fraction, which showed a reduced ability to synthesize acid phosphatase under leucine selection due to their inefficient leucine complementation. Otherwise, the cells from the top fraction behaved like normal growing cells (e.g., cells under uracil selection) in secreting acid phosphatase into the periplasmic space and the medium, while cells from the bottom fraction did not grow, accumulated acid phosphatase intracellularly, and clearly showed the phenotype of cells grown under leucine selection (Fig. 16). Dense cellsin

bottom -leucine

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Flgure 16 Radiolabeling and subfractionation of acid phosphatase with dense cells enriched in thebottom fraction of a Ludox gradient. YB18/pDP34/GAPFLpH05 transformants were grownunder uracil and leucine selectionconditionsas described in Fig. 13. After 6 h of fermentation the cells were harvested and applied to 55% Ludox-AM (DuPont) gradient solutions. After centrifugation (22.OOOg at 10°Cfor U)min) the cells in the bottom fraction of the gradients were collected. Pulse labeling inthe absence of uracil (-uracil)and leucine (-leucine), respectively, and subfractionation was performed as described in Fig. 15. Subfraction m (lanes 1 and 4) represents extracellular acid phosphatase,fraction p (lanes 2 and 5) periplasmic enzyme, and fraction i (lanes 3 and 6 ) intracellular acid phosphatase.

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the bottom fraction of the gradient had the same properties, whether they originated from a culture under uracil or leucine selection (Fig. 16). 6.4

Discussion

High-level expression of a protein in yeast isoften achieved by using 2pm-based expression vectors. Characteristicfeatures of this type of vector are the high average copy number, but also a rather broad copy number distribution within the cell population due to unequal partitioning (13). We tried to establish a link between gene dosage and the behavior of subpopulations of cells and to make this visible in our system. Under uracil selection,cells with evena low plasmid copy number can grow (efficient complementation). They synthesize and secrete mature acid phosphatase, as expected. Under leucine selection, however, cells with a low plasmid copy number are starved of leucing, dueto the poorly expressed leu2-d allele. Cells with a high copy number produce enough, leucine for growth, but concomitantly, the high gene dosage for acid phosphatase leads to an intracellular buildup of acid phosphatase. This, in turn, results in a block-for reasons not yet understood-of vectorial transport to the bud and an arrest of growth. Changes inthe actin staining pattern indicate changes in the cytoskeleton, which can be correlated to defects in vectorial secretory transport and bud formation (177). Continued synthesis of acid phosphatase leadsto intracellular accumulation of slightly overglycosylated enzymeat a late stage inthe secretory pathway. Part of this overglycosylated acid phosphatase reaches the periplasmic space probably in an undirected, random fashion by fusion of organelles with the plasma membrane. In summary, therefore, under leucine selection, we see two overlapping effects. Leucine starvation for one subpopulation of cells and overproduction of acid phosphatase for another subpopulation of cells resulting in the nongrowth phenotype of the culture. Interestingly, a similar subpopulation of cells with a high plasmid copy number and intracellular accumulationof acid phosphataseis also present under uracil selectionbut is hidden by the majority of normally growing cells in the culture. Dense cells with accumulated acid phosphatase are enriched in thebottom fraction of Ludox density gradients. The dense cells from cultures under uracil or leucine selection behave virtually identically, and the properties of these cellsare very similar to those determinedfor a cell culture under leucine selection as a whole. We have shown that in an overexpression situation for a homologous yeast protein, such as acid phosphatase, a subpopulation of cells has a

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nongrowth phenotype with aberrant intracellular accumulation of the protein. Under leucine selectionthis phenotype dominates and is therefore easily detectable, whereas it is not apparent under uracil selection of acid phosphatase observed conditions. The intracellular accumulation supports the ideathat even for a homologous yeastprotein, the secretion capacity of the cell can be limiting under certain conditions.

7

DISULFIDE-LINKED DIMERIZATION OF RECOMBINANT HUMAN INSULIN-LIKE GROWTH FACTOR-1 RELATED TO ITS FOLDING IN THE YEAST ENDOPLASMIC RETICULUM

7.1 Introduction

Human insulin-like growth factor-l (IGFl) is a single-chain, 70-amino acid polypeptide which shares a high degree of homology withthe twochain insulin molecule (178). Like insulin, IGFl has three intramolecular disulfide bonds.IGFl is of pharmaceutical interest because clinical studies indicate its potential use inthe treatment of someforms of dwarfism and diabetes,and also in hastening the process of wound healing (178). Active monomericIGFl has been produced as product a secreted from the yeast Saccharomyces cerevzkiae.Analysis of the IGF1-like molecules in the yeast culturesupernatant on immune-blots reveals that the secreted molecules are predominantly disulfide-bonded dimers. We have addressed the possibilitythat misfolding of the nascent IGFl polypeptide maybe a reason for dimer formation.

7.2 Protein Folding: The Role of Signal and Leader Peptides Most proteins destinedfor secretion require transient amino-terminal sequences, containing15 to 30 amino acids,for targeting to the endoplasmic reticulum (ER) (179). These sequences, termed signal peptides, mediate translocation of newly synthesized proteins across the membraneof the ER (180). It is believed that the initial foldingof nascent polypeptides is delayed by the presenceof signal sequencesat the N-terminus (181).This delay in foldingis thought to permit a translocation competent state (181, 182). Upontranslocation, the signal peptide is usually cleaved by the enzyme signal peptidase, and then the protein is free to acquire its native folded conformation in the lumen of the ER (183). Secretion of heterologous proteins has been achieved from S. cerevisiae (91) by fusingthe signal sequences of yeast secretory proteins to the coding

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regions of various polypeptides. Proteins have been secreted utilizing the 17-amino acid phosphatase signal sequence encoded by the PH05 .gene (184), the 19-amino acid invertase signal sequence encoded by the SUC2 gene (185), and the 85-amino acid preprosequence of the prepro-a-factor encoded by the MFa gene (186). The preprosequence of the prepro-afactor, usually referred to as the a-factor leader (aFL), is the signal quite often used for the efficient secretion of foreign proteins (187). The aFL is an unusually long secretion signal consisting of a classical 19-amino acid signal or presequence and a 66-amino acid proregion (pro-aFL). The signal sequence segment of the aFL has been used for membrane targeting and has been shown to be cleaved during translocation (79,188). However, the function of the proregion, which is processed only later in the transGolgi by the product of the KEX2 gene (189), in the secretion and folding of heterologous proteins is not clearly understood.

7.3 Secretion of Recombinant Insulin-Like Growth Factor-1 IGFl is secreted from yeast using an a-factor leader-IGF1 gene fusion (153). The IGFl expression cassette consists of a 400-bp fragment of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter and a 275-bp fragment from the MFa gene which is used for termination of transcription (153,190). It was observed that the signal peptides from the PH0.5, SUC2, and MFa genes (i.e., PHOSss, Invss, and aFss, respectively) do not allow the secretion of recombinant IGFl from yeast (190). It appears that 17- to 19-amino-acid-long classical secretion signals are not sufficient for the translocation of IGFl across the ER. Being the first step in the secretion pathway (191), the translocation event would make possible the secretion of IGFl into the yeast culture medium. Surprisingly, IGFl is secreted only by leader peptides (i.e., PHOSssproaFL, InvssproaFL, and aFL), where each of the three signal peptides, PHOSss, Invss, and aFss, are fused to the 66-amino acid pro-aFL sequence (190). This implies that the proregion of the aFL is essential for the translocation of IGFl . With only signal peptides present at the N-terminus, IGFl probable folds too quickly in the cytoplasm, acquiring a conformation that is translocation incompetent. It is likely that covalent attachment of the pro-aFL to IGFl causes a delay in the folding of IGF1, which confers the optimal conformation crucial for translocation (181,182).

7.4

Disulfide-Linked Dimerization of IGF1 Related to Folding

The majority of the IGFl molecules secreted from yeast using any of the three leader peptides are not active monomers but inactive disulfide-linked

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dimers. It has been suggested that dimer formation could be the result of a specific charged interaction between two IGFl molecules during the process of folding in the yeast cell (192). It can also be argued that after permitting translocation, the covalently linked pro-aFL has a deleterious effect on the folding of IGFl . The proregion is removed in the late Golgi only after folding has occurred. Processing takes place when the membrane-bound endoprotease Kex2p cleaves at a pair of exposed dibasic amino acids linking the N-terminal pro-aFL to the C-terminal IGFl . Therefore, it is possible that the presence of the extraneous pro-aFL sequence during folding in the lumen of the ER could cause a distortion in the folding pathway of IGFl (193), which may result in the formation of dimers.

7.4.I

Mutant Kex2 Enzyme Function in the Endoplasmic Reticulum as a Tool to Study Formation of IGFI Dimers To investigate whether pro-aFL is really involved in IGFl dimnerization, we have examined the effect of a Kex2p variant, soluble Kex2pHDEL (sKex2pHDEL), which could function in the ER (194). This mutant enzyme, if active, should remove the proregion from the precursor protein in the lumen of the ER rather than in the late Golgi. The early processing would diminish the effect of the proregion of the aFL on the folding of IGFl and enable the mature IGFl molecule to fold independently. 7.4.2 C-Terminal His-Asp-Glu-Leu Sequence Allowing Retention of a Soluble Form of the Kex2 Enzyme in the Endoplasmic Reticulum It has been reported that a soluble form of the Kex2 enzyme (sKex2p) (195) is obtained by deleting the C-terminal 200-amino acids from the wtKex2p (196). The deleted region includes the membrane-spanning region of the enzyme. The sKex2p variant has been shown to be secreted in considerable amounts into the yeast culture medium when expressed on a multicopy plasmid (195). No Kex2p-like endoproteolytic activity (102) is observed in the medium when wtKex2p is expressed similarly. Secretion of sKex2p is appreciably lower if the S. cerevisiae ER-retention signal HisAsp-Glu-Leu (HDEL) (197) is attached to the C-terminal end of the soluble form of Kex2p (unpublished observations). To answer the question as to whether the HDEL sequence bestows the property of ER retention on sKex2p, we have used, as in vivo substrates, two precursor IGFl molecules that accumulate in the ER. The enzyme that functions in the ER should generate mature molecules from an ER-accumulated reservoir of intermediates. When the wtaFL sequence is used for secretion, mature IGFl is the major intracellular entity. Through pulse-chase experiments (153) it has

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been shown that maturation of IGFl occurs via intermediatesthat bear Asn-linked core sugars (198). Sincethe proregion of the (YFL,normally removed in the late Golgi, has three sequons for N-glycosylation (AsnXaa-SerIThr), the nature of the oligosaccharides attached to the proaFL can actas convenient tagsto monitor the location of precursor intermediates in the cell (199). The precursors residing in the ER bear only core sugars, which are observed as distinct bands after electrophoresis on polyacrylamide gels. Intermediatesthat have traveled beyondthe ER have a mixture of outer-chain mannosesand are identified by slower gel mobility (i.e., larger molecular mass)and diffuse bandson gels. Two mutated aFL sequences (Mut2 and Dell; Tables 5 and 6 ) fused to IGFl cause intracellular accumulationof precursor IGFl molecules (194). Only a very small amount of the precursors undergoesmaturation. These molecules still have the proregion of the aFL attached to IGF1. Mostly N-linked core sugars are present in these intermediates (Fig. 17), typical of molecules that have not traveled beyond the ER in the secretion pathway (191, 198). If the HDEL tetrapeptide allows retentionof sKex2p in the ER, then among the three Kex2p variants, sKex2pHDEL should be unique in releasing mature IGFl from an intracellular pool of precursors which accumulates in the ER. The three Kex2p variants, wtKex2p, sKex2p, and sKex2pHDEL, have been subcloned on the multicopy plasmid pDP34B S. cerevisiae 2-pm sequence (118). (23,194), which contains the complete The plasmids not only encode one of the Kex2p variants but also carry the IGFl expression cassettes containing the mutated aFL sequences (Table 5). Appropriate plasmids (Table 6) were transformed in a yeast strain, AB1 lOkex2, wherethe genomic copy ofthe KEX2 gene wasdisrupted by gene replacement, and no functional wtKex2p is expressed (194). Yeast transformants were grown in an uracil-selective mediumfor expression

Table 5 Specific Amino Acid Changes in the Mutated aFL Mut2 and a13-AminoAcidDeletionInvolvingthe First Glycosylation Site (Dell) in the aFL (194)

Deletion Mutated aFL sequence Mut2 Dell

Mutation Ala20 to Asp20 and Pro21 to Leu21

-

Pro21 to Ile33

Table 6 Plasmids Used for Expression of IGFP

Expression cassette(s) coding for:

aFL sequence Yeast Plasmid used

strain@)used for transformation ~~

~~

IGFl ABllO and ABllOkex2 WT pBC23 IGFl AB1 10 Mut2 pBCl2 IGFl ABllO pBC13 Del 1 IGFl and wtKex2p pBC17 Mut2 AB1 lOkex2 IGFl and sKex2p ABllOkex2 pBC18 Mut2 IGFl and sKex2pHDEL ABllOkex2 pBC19 Mut2 IGFl and wtKex2p ABllOkexZ pBC20 Del 1 IGFl and sKex2p AB1 lOkex2 pBC211 Del IGFl and sKex2pHDEL ABllOkexZ pBC22 Del 1 IGFl and wtKex2p ABllOkex2 WT pBC24 IGFl and sKex2p AB1 lOkex2 WT pBC25 IGFl and sKexwpHDEL AB1 lOkex2 WT pBC26 Vhe vector pDP34B either bears only the IGFl expression cassette (153) containing one of the aFL secretion signals, or both the IGFl expression cassette and one of the three KEX2 variants. Expressionof IGFl is under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter (153).

Il Figure 17 Endoglycosidase F (Endo F)-digested products (lanes 2,4,6) of intracellular IGF1-likeproteins from AB1 10transformants harboring IGFl expression plasmids pBCl2 and pBC13 (Table 6) bearing the mutated aFL sequences (Table 5). All cell lysates were from yeast cultures (153) grown for 72 h.After completion of digest(l%), 0.5 ml of Laemmli buffer was added. A f t e r separation ona SD%15% polyacrylamide gel, proteins were blotted onto poly(vinylidene diflouride) membrane (Millipore) for Western blot analysis. Transferred proteins were detected with anti-IGF1 polyclonal antiserum (153).Lanes 1 and 2, unglycosylated proteins from Mutl (194), which accumulate inthe cytoplasm (asa control for Endo F digest); lanes 3 and 4, Mut2; lanes5 and 6, Dell; lane 7 , 150 ngof HPLC-purified IGFl monomer. The 17-, 27-, 39-, and 50-kDa bands used as markers belong to the prestained low-range standard proteins (Bio-Rad); M, IGFl monomer.

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Iry

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sKex2p hdei

Figure 18 In vivo comparison of Keep, sKex2p, and sKedpHDEL using as substrates intracellular entities accumulated in the ER (194). IGF1-like proteins from cells grown from ABllOkex2 transformants bearing plasmids (A) pBC17, pBC18, and pBC19; and (B) pBC20, pBC21, and pBC22. All plasmids are listed in Table6. Two transformants from each the of six transformations were used for the Western blot analysis. Proteins from 2 pL cell lysates were reduced with dithiothreitol and were separated by 15% SDS-PAGE. After blotting (as in Fig. 17), IGF1-like proteins were detected with the anti-IGF1 antiserum. The markers used were the same as in Fig. 17.

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of IGFl (153). Harvested cells were lyzed and analyzed by Westernblotting (194). Comparison of the intracellular proteins (Fig. 18) clearly shows that sKexpHDEL is indeed distinct from the other two variants. In contrast to wtKex2p and sKex2p, sKex2pHDEL does form more mature IGFl molecules fromthe intracellular precursor intermediates accumulated in at the C-terminus the ER.This strongly indicatesthat the HDEL sequence of sKex2p has allowed a partial retention of sKex2p in the ER.

7.4.3 Use of sKex2pHDEL to Prevent Intermolecular Disulfide-Linked Dimerization of lGF7 Only 10 to 15% ofthe secreted IGFl molecules are monomeric usingthe wtaFL as a leader peptide (153,190).A large proportion of the total secreted product is dimeric (192,200). Knowing that the C-terminal HDEL tetrapeptide may allow retention of a soluble form of Kex2pthe in endoplasmic reticulum, we have attempted to remove the proregion of the wtcuFL in the lumen of the ER. Plasmids pBC24, pBC25, and pBC26 (Table 6)were transformed in the yeast strain AB1 lOkex2,and supernatants from yeast cultures were compared under nonreducingand reducing conditions (Fig. 19). It appears that dimer formationis scarcely observedon analysis of IGF1-like molecules secretedfrom strains transformed with the plasmid pBC26 and carrying the sKex2pHDEL and IGFl expression cassettes. This indicatesthat the proregionof aFL, after permitting translocation, does influencethe folding of IGFl in a way that facilitates dimer formation (201). 8 CONCLUSION The Kex2 endoprotease can be mislocated from the Golgi to the ER using the S. cerevisiae ER-retention signal HDEL.The proregion of the aFL, which is essentialfor the translocationof IGFl, can be cleaved inthe ER using the ER-retained variant of the Kex2 enzyme. Removal ofthe proregion early inthe secretion pathway hasan impact on the folding of the nascent IGFl polypeptide. It prevents the formation of disulfide-linked IGFl dimers. It is possiblethat sKex2pHDEL could be usedas an in vivo biochemical tool to study the broad role of proregions in the folding and intracellular transport of other precursor proteins. ACKNOWLEDGMENTS Without the helpof many of our colleagues, this chapter would not have been possible. We greatly appreciate theirsupport in providing original

uuu KexPp sKex2p sKex2p

HDEL

Figure 19 Comparison of IGF1-like proteins secreted from ABllOkex2 transformants bearing expression plasmids pBC24, pBC25, and pBC26 (Table 6) on a Western blot (as in Fig. 17). Two transformants from each of the three transformants were usedfor analyses. An AB1 10 transformant of plasmid pBC23was used as a control. Two milliliters of supernatants was concentrated using Centricon-3 filters (Amicon). Supernatants from pBC23 and pBC24 were concentrated twofold and the ones from pBC25 and pBC26 were concentrated fourfold (201). Proteins from 5 pL of concentrated supernatants were analyzed, as in Fig. 17. (A) Lane 1, 150ng of purified IGFl monomer (M); lane 2, pBC23::ABllO; lanes 3 and 4, pBCM:ABllOkex2; lanes 5 and 6, pBC25::ABllOkex2; lanes 7 and 8 , pBC26::ABl lOkex2. Rainbow-colored (Amersham) protein markers (low molecular weight range) were used as standards. (B) Sameas in (A), exceptthat the supernatants were treated with dithiothreitol.

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data and their scientific input. Many thanks to P. Fiirst, H. Grossenbacher, R. Kleene, W. Mtirki, C. Stephan, A. Strauss, K. Takabayashi, and H. J. Treichler.

REFERENCES 1. Tuite MF. Expression of heterologous genes. In: Tuite MF, Oliver SG, eds. Saccharomyces. New York: Plenum Press, 1991:169-212. 2. Barr PJ, Brake AJ, Valemela, P. eds. Yeast genetic engineering. Boston: Butterworth, 1989. 3. Guthrie C,Fink GR, eds. Methods in Enzymology, Vol.194. Guide to Yeast Genetics and Molecular Biology. San Diego: Academic Press, 1991. 4. Strathern JN,Jones EW, BroachJR, eds. The molecular biology ofthe yeast

Saccharomyces. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1981. 5. Rose AH, HarrisonJS, eds. The Yeasts, Vol. 3. San Diego: Academic Press, 1989. 6. Hinnen A, Hicks JB, Fink CR. Transformation of yeast. Proc Natl Acad Sci USA 1978; 75:1929-1933. 7. Rothstein RJ. One-step gene disruption in yeast. Methods Enzymol 1983; 101:202-211. 8. Winston F, Chumley F, Fink

CR. Eviction and transplacement of mutant genes in yeast. Methods Enzymol 1983; 101:211-228. 9. Orr-Weaver TL, Szostak JW, Rothstein RJ. Genetic applications of yeast transformation with linear and gapped plasmids. Methods Enzymol 1983; 101~228-245. 10. Boeke JD, Xu H, Fink GR. A general method for chromosomal amplification of genes in yeast. Science 1988; 239:280-282. 11. Beggs JD. Transformation of yeast by a replicating hybrid plasmid. Nature 1978;275:104-109. 12. Rose AB, Broach JR. Propagation and expression of cloned genes in yeast: 2pm circle-based vectors. Methods Enzymol 1990; 185:234-279. of Saccharomycesmrevisiue. Yeast 1988; 13. Futcher AB. Thecircleplasmid 4:27-40. 14. Janes M, Meyhack B, Zimmermann W, Hinnen A. The influence of GAP

promoter variants on hirudin production, average plasmid copy numberand cell growth in Saccharomyces cerevisiue. Curr Genet 1990; 18:97-103. 15. Erhart E, Hollenberg CP. The presence of a defective LEU2 gene on a 2p DNA recombinant plasmid of Succhuromyces cerevisiue is responsible for curing and high copy number. J Bacteriol 1983; 156:625-635. 16. Unternahrer S, Pridmore D, Hinnen A. A new system to amplify 2p plasmid copy number in Saccharomyces cerevisiae.Mol Microbiol 1991; 5:1539-1548. 17. Hartwell LH, Mortimer RK, Culotti J, Culotti M. Genetic control of the cell division cyclein yeast. V. Genetic analysis ofc& mutants. Genetics 1973; 74~267-286.

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A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor proteins. Nature 1988; 332:800-805. 183. Freedman RB. Protein disulfide isomerase: Multiple roles in the modification of nascent secretory proteins. Cell 1989; 57:1069-1072. 184. Meyhack B, Bajwa W, Rudolph H, Hinnen A. Two yeast acid phosphatase structural genes are the result of a tandem duplication and showdifferent degrees of homology in their promoter and coding region. EMBO J 1982; 1:675-680. 185. Taussig R, Carlson M. Nucleotide sequence of the WC2 gene for invertase. Nucleic Acids Res 1983;11:1943-1954. 186. Kurjan J, Herskowitz I. Structure of a yeast pheromone gene (MFa): A

187.

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putative a-factor precursor contains four tandem copies of maturea-factor. Cell 1982;30:933-943. Brake AJ. Secretion of heterologousproteins directed by the yeast a-factor leader. In: Barr PJ, Brake AJ, Valenzuela P, eds. Yeast Genetic Engineering. Boston: Butterworth, 1989:269-280. Waters MG, Evans EA, Blobel G. Preprw-factor has a cleavable sequence. J Biol Chem 1988; 263:6209-6214. Fuller RS, Sterne RE, Thorner J. Enzymes required for yeast prohormone processing. Annu Rev Physiol 1988; 50:345-362. Chaudhuri B, Steube K, Stephan C. The proregion of the yeast prepro-afactor is essential for membrane translocation of human insulin-like growth factor-l in vivo. Eur J Biochem 1992; 206:793-800. Rothman JE, Orci L. Molecular dissection of the secretion pathway. Nature 1992; 355:409-415. Chaudhuri B, Helliwell S, Priestle JP. A L y ~ ~ ~ - t o - Gmutation lu*~ in the human insulin-like growth factor-l prevents disulfide linked dimerisation and allows secretion of BiP when expressed in yeast. FEBS Lett 1991; 294:

213-216. 193. Hober S, Forsberg G, Palm G, Hartmanis M,Nilsson B. Disulfide exchange folding of insulin-like growth factor I. Biochemistry 1992; 31:1749-1756. 194. Chaudhuri B, Latham SE, Helliwell SB, Seeboth PG. A novel Kex2 enzyme

can process the proregion of the yeast a-factor leader in the endoplasmic reticulum instead of in the Golgi. Biochem Biophys Res Commun 1992; 183~212-219. 195. Seeboth PG, Heim J. In-vitro processing of yeast a-factor leader fusion

proteins using a soluble yscF (Kea) variant. Appl Microbiol Biotechnol 1991; 35:771-776. 1%.

Mizuno K, Nakamura T, Ohshima T, Tanaka S, Matsuo H. Yeast KEX2 gene encodes an endopeptidase homologous to subtilisin-like serine proteases. Biochem Biophys Res Commun 1988; 156:246-254.

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197. Pelham HR. Control of protein exit from the endoplasmic reticulum. Annu Rev Cell Biol 1989; 5:l-23. 198. HirschbergCB,SniderMD.Topographyofglycosylation in the rough endoplasmic reticulum and Golgi apparatus.Annu Rev Biochem 1987; 56: 63-87. 199. Graham TR, Emr SD. Compartmental organization of Golgi-specificprotein modification and vacuolar protein sorting events defined in a yeast sed8 ( N W ) mutant. J Cell Biol 1991; 114:207-218. 200. Elliott S, Fagin KD, Narhi LO, Miller JA, Jones M, Koski R, Peters M, Hsieh P, Sachdev R, RosenfeldRD, Rohde MF, Arakawa T. Yeast-derived recombinanthumaninsulin-likegrowth factor I: Production, purification and structural characterization. J Protein Chem 1990; 9:95-104. 201. Chaudhuri B, Stephan C. A modified Kex2 enzyme retained in the endoplasmic reticulum prevents disulfide-linked dimerisation of recombinant human insulin-like growth factor-l secreted from yeast. FEBS Lett 1992; 304:41-45.

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Gene Expression in Methylotrophic Yeasts Gerd Gellissen Rhein Biotech GmbH, Diisseldotf, Germany

Cornelis P. Hollenberg Heinrich-Heine University, Diisseldorf, Germany

Zbigniew A. Janowicz Rhein Biotech GmbH, Dusseldotf, Germany

1 INTRODUCTION The development of several microorganisms and eukaryotic cell lines as hosts for heterologous gene expression has led to an increasing number of options for the production of recombinant proteins. The initial systems, the bacterium Escherichia coli (1,2) and the yeast Saccharomyces cerevisiae (3,4), respectively, are now supplemented by an increasing number of alternatives (5-8), of which some examples are described elsewhere in this volume. For industrial applications the system of choice should produce an optimal amount of authentic bioactive material in a short time in a reproducible and stable production process. Therefore, expression systems have to fullfil several prerequisites to allow such an efficient and economical production of a recombinant protein. The cells should be robust and easy to handle, and they should grow to high cell densities on low-cost media containing cheap energy and carbon sources. Adequate expression vectors must be available and the host must be capable of synthesizing the desired protein either intracellularly or, if certain eukaryotic modifications are required, extracellularly. If it fails to assure a correct fold195

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ing, laborious and cost-intensive in vitro refolding and modification processes might have to be applied. As unicellular eukaryotic organisms, yeasts combine the ease of genetic manipulation with the ability to perform eukaryotic processing steps on the polypeptides expressed. When the traditional S. cerevisiae systemwas used for heterologous gene expression, disadvantages such as strain instability during the production process, low productivity, and an inappropriate glycosylation were encountered in several cases. Therefore, a search was initiatedto find alternative non-Saccharomyces yeasts suitable as hosts for recombinant protein production. Two facultative methylotrophic yeasts, Hansenula polymorpha (9-12) and Pichia pastoris (13, 14), have been developed duringthe recent past. These two ascomycetes are characterized by efficient utilization of methanol as the sole energy and carbon source. The ability to grow on this C, carbon compound is found in a variety of bacteria (15-16) and a limited number of filamentous fungi and yeasts belonging to the genera Candida, Torulopsis, and Hansenula and Pichia, on which we focus (19-22). These yeasts share a general pathwayby which, on the one hand, methanol is metabolized to carbon dioxideto generate energyand is used, on the other hand, for assimilation to produce biomass. In these organisms growth on methanol is accompanied by a strong induction of enzymes involved in methanol metabolism. Genes encoding key enzymes of methanol have been isolated and characterized (23-27). A s a consequence, their strong inducible promoters became availableas components for the expression of foreign genes. Techniques were developedto introduce heterologousDNA into suitable methylotrophic hosts, and expression systems were established for heterologous gene expression whichare characterized by high stability, viability, and productivity. In the following section the expression systems based on these two methylotrophs are described and discussed.

2 METHANOL METABOLISM AND ITS REGULATION IN METHYLOTROPHIC YEASTS The complex methanol utilization pathway shared by the methylotrophic yeasts is highly compartmentalized. The initial reactions take place in specialized microbodies, peroxisomes, followed by subsequent metabolic steps in the cytoplasm (19,27-30). Methanol enters the peroxisomesand is then oxidizedby a specific methanol (alcohol) oxidase (MOX or AOX) to generate formaldehyde and hydrogen peroxide (19,22,28,30-37). Methanol (alcohol) oxidases isolated from different yeast species exhibit similar

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properties. Theenzyme contains eight subunits yieldinga protein of Some 600 kDa (30,34). It is a flavoprotein and contains one FAD moiety per subunit. Theenzymes oxidizeshort-chain (C,to C,) aliphatic primary alcohols. V , , decreases and K,,, increases with increasing chain length. V,, values reported for methanol range between3 and 20 pA4 m i d (mg between 0.2 and 2.0 mM.The K,,, value of the enzyme protein)", the K,,, for formaldehyde, which can serve as a substrate in its hydrated form, value for oxygen of the H. polymorpha is 3 to 10 times higher. The K,,, enzyme lies between0.24 and 0.4 mMmolecular oxygen, dependingon the methanol concentration present in the system (36,37). This low affinity for oxygen forces the enzyme to work at suboptimal oxygen concentrations in the cell (oxygen concentration in air-saturated is water about of the enzyme for its 0.2 mM).This adds to the poor catalytic properties physiological function under in vivo conditions. This deficiency is compensated by a massive production of the protein under conditionsof enzyme requirement (see below). The toxic hydrogen peroxide generated in the oxidase reactionis decomposedto water and molecular oxygen by a peroxisomal catalase (19,35,37,38). The catalase activity is high during growth on methanol. In H. polymorpha, specific activities of 1.45 mM m i d (mg protein)' have been reported, thus being l@ and 104 times in excess of the oxidase (28).MOX and CAT activitieswere found to be coregulated to some degree (19) (see also Section 3). Formaldehyde, generated bythe oxidase reaction, enters either the cytosolic dissimilatory pathway to yield energy or the assimilatory pathway to participate inthe generation of biomass, which starts with a first step of a peroxisomal localization. This first step, a key reaction of the assimilatory pathway,is catalyzed bythe peroxisomal enzyme dihydroxyacetone synthase, yielding dihydroxyacetone and glyceraldehyde-3-phosphate in a transketolase reaction between formaldehyde and xylulose-5-phosphate (39-43). These C, compounds are further assimilated in the cytosol. Dihydroxyacetone is phosphorylated by a dihydroxyacetone kinase.Catalyzed by an aldolase, it reacts subsequently with glyceraldehyde to form fructose-l,6-biphosphate. By the action of a phosphatase, fructose-6phosphate is formed. In several intermediate steps xylulose-5-phosphate is eventually regenerated by transaldolase, transketolase, pentose phosphate isomerase, and epimerase reactions ina cyclic pathway. One-third of the glyceraldehyde-3-phosphate generated is converted into biomass by standard reactions of the central cell metabolism (21,22,28,30). Some of the formaldehyde produced in the methanol oxidase reaction enters the cytosol, where itis catabolized in two subsequent dehydrogenase reactions (44-47). aInfirst step, formaldehydeis oxidizedto formate

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by a formaldehyde dehydrogenase(48-50), formate is subsequently oxidized to carbon dioxide by formate dehydrogenase (49,50). Again, formaldehyde dehydrogenase is similar in all yeasts analyzed so far (30,4850). The enzyme is strictly dependent on glutathione for activity. It does not use free formaldehyde but the thiohemiacetal formed from reduced glutathione and formaldehyde as substrate in a NAD+-dependent reaction to produce S-formylglutathione. S-Formylglutathioneseems to be the substrate of the subsequent formate dehydrogenase reaction in H. polymorpha, since this enzyme has a 40-fold-higher affinity for this compound than for formate (51). The enzyme hydrolyzes S-formylglutathione in the presence of NAD+. Therefore, it is assumedthat a first hydrolysis leads to an enzyme-bound formate which is oxidized in a second step to carbon dioxide. In the methylotrophic yeast speciesKfoeckera sp. 2201 and Candida boidinii, two distinct enzymes have been identified for this process. An initial hydrolysis to glutathione and formate by Sformylglutathione hydrolases (52,53) is followed by formation of carbon dioxide from the formate generated by formate dehydrogenase activity (30). A scheme of this utilization pathway, which has been reviewed comprehensively (30,54), is presented in Fig.1. There might be additional possibilities to yield energy during growth on methanol. Sybirnyet al. (55) isolated H. polyrnorpha mutant strains that are deficient in formaldehyde/formate dehydrogenase. These mutants were found to grow on methanol as a sole energy and carbon source, indicating that enz$nes of direct formaldehyde catabolism are not indispensable for methylotrophic growth. Since inhibition of the tricarboxylic acid cycle results in a growth repression, the authors suggest that dissimilation of methanol and the supply of energy during methylotrophic growth is achieved primarily by oxidation of C, compounds generated by the DHAS transketolase reaction viathe tricarboxylic acidcycle. According to this concept, the direct cytosolic oxidation serves mainly for detoxification of formaldehyde. Growth on different carbon sources results in distinct patterns of intracellular H. polyrnorpha proteins. In methanol-grown cells three prominent polypeptidesare present in large amounts. These abundant proteins are key enzymesof the methanol metabolism described before: methanol oxidase (MOX), formate dehydrogenase (FMDH), and dihydroxyacetone and FMDH canalso be desynthase (DHAS). Significant levels MOX of tected in glycerol-grown cells, but they are absent when glucoseused is as a carbon source (Fig. 2). Thus synthesis of these key enzymes is subject to a repression/derepression/induction mechanism forcedby the carbon

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DHA

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Cytosol Figure 1 Methanol utilization pathway and its compartmentalization in H. polymorphs. Methanol is oxidized within the peroxisome by methanol oxidase (MOX) to generate formaldehyde and hydrogen peroxide (1). The toxic hydrogen peroxide is metabolized to water and oxygen (2). To some extent the formaldehyde generated enters the cytosol to be dissimilated to carbon dioxide by two subsequent dehydrogenase reactions, catalyzed by formaldehyde dehydrogenase (3) and formate dehydrogenase (4) (FMDH). For assimilation, the formaldehyde remaining in the peroxisome reacts with xylulose-5-phosphate (Xu,P) by the action of dihydroxyacetone synthase (DHAS) (5). The resulting C, compounds glyceraldehyde 3-phosphate (GAP)and dihydroxyacetone @HA) enter the cytosol. The DHA is phosphorylated by a dihydroxyacetone kinase (6) to dihydroxyacetone phosphate (DHAP). DHAP and GAP produce fructose l,&biphosphate (FBP) in a reaction catalyzed by fructose 1,6-biphosphate aldolase (7). Subsequently, FBP is dephosphorylatedby fructose 1,5-biphosphatase (8). In further steps X h P is regenerated. One-third of the GAP molecules produced enter the central metabolism of the cell to generate biomass. The H. polymorpha genes isolatedand characterized for methanol oxidase (MOX), formate dehydrogenase (FMD), dihydroxyacetone synthase (DAS), and catalase (CA'I) are indicated in the pathway at the position of the enzymes they are coding for. In Pichia pastoris, two highly homologous alcohol oxidase genes designated AOXl andAOX2 have been isolated and characterized. (From Ref. 42; figure and legend modified according to Refs. 22 and 144).

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A

B

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Figure 2 SDS-PAGE analysis of intracellular H. polymorpha proteins. Crude extract from cells grown in media supplemented withdifferent carbon compounds (lane A, medium containing 2% glucose; lane B, 0.5% glycerol; and lane C, 1% methanol) were separated by SDS-PAGE through 12.5% gels and visualized by Coomassie Blue staining. The synthesis of key enzymes of the methanol utilization pathway is repressed in glucose-containing media, derepressed in glycerolcontaining media, and induced in methanolcontaining media. Key enzymes are dihydroxyacetone synthase@HAS; M, = 78,000), methanol oxidase (MOX; M, = 69,000), and formate dehydrogenase (FMDH; M, = 38,000).

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compounds of the culture medium (56,57). In P. pastoris, substantial amounts are visible only in methanol-grown cells, although low concentrations of alcohol oxidase (2%of the inducedstatus) during growth under glycerol-limited conditions have been reported. Thus expression of these proteins follows a repression/induction mechanism in contrast to the situation in Hansenula (58). Upon addition of methanol, the respective genes are heavily induced (see the next section) and a massive proliferation of the peroxisomes, the compartment for MOX, DHAS, and the catalase (CAT), is promoted (59-61). The methanol oxidaseis an octamer forming a crystalline lattice that constitutes the peroxisomal skeleton (62-64) containing DHAS, CAT, and furtherenzymes within. In recombinantH. polyrnorpha strains overproducing methanol oxidase, formation of irregular giant peroxisomes densily packed with huge enzyme crystalloids were observed. In these transformants, activeMOX can constitute upto 70% of the total cell protein (65). Surprisingly, this crystalline lattice can also be formed in the cytoplasm of peroxisome-deficient (per)mutants of H. polyrnorpha. Thus its formation is independent of a peroxisomal location (66). In methanol-grown continuous cultures of H. pofymorpha,MOX constitutes about 20 to 30% of total cell proteins; FMDHand DHAS, 10 to 20%. Huge peroxisomes can take up to 80% of the cell volume (51; see also Fig. 2). In cells grown ona repressive carbon source such as glucose or ethanol, the typical peroxisomal structures cannot be detected. Instead, a few small microbodies functioning as glycosomes are observed, from which peroxisomes proliferate after transfer into media with inductive conditions (67-70). Yeast peroxisome proliferation and growth in response to the environment are controlled by a variety of different genes (71). First steps ina genetic approach to understanding the complex biogenesis of this organelle have been undertaken in isolating peroxisomedeficient or peroxisome assembly mutants in S. cerevisiae (71,72), H. polyrnorpha (73,74), and P. pastoris (75). Back transfer of induced cells into media witha repressive carbon source results in rapid degradation of peroxisomal enzymes and peroxisomes by an autophagic process(68-70). This process is initiatedby formation of a 2- to 12-layered membrane enclosing the organelle. A vacuole becomes associated with this structure and is eventually incorporatedto release hydrolytic enzymes. Theabundant methanol oxidaseis rapidly degraded (76-79). Since a rapid degradationof the enzymes isnot observed in the cytosolic crystalloids per in mutants, it is believedthat the mechanism triggering the degradative inactivationof methanol metabolism enzymes is not directed against the protein itself but against the organelle

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harboring it(66).Additional inactivation occurs by protein modifications, resulting inthe release of the prosthetic FAD,which is indispensablefor enzyme activity (68). The peroxisomaland cytosolic methanol utilization enzymes are regulated in a coordinated fashion. This is similarly found for all methylotrophic yeasts, but the mechanisms are not identical, as already pointed out. The peroxisomal MOX and cytosolic FMDH are regulated by the catabolite repression/derepression/induction mechanism described earlier (inPichia by the repression/induction mechanism).It is not clear whether methanol itself or another metabolic intermediate acts as the inducing agent. In Hansenula, induction with methanol can also be considered as a strong derepressionby the absence of repressivecarbon sources such as glucose or ethanol. Accordingly, the inductive effect be canoverruled by the high glucose orethanol concentration present inthe system. The derepression effect is concentration dependent. At high concentrations of a derepressive carbon source suchas glycerol (2070) in the culture medium, only a weak synthesis of the methanol-inducible enzymes is observed, which increases at lower concentrations (0.2 to 0.4% glycerol). Consequently, a slight derepression can be detected in low concentrations of a repressive carbon compound such as .glucose(76). The expressionof D M , the peroxisomal assimilatory pathway enzyme, and of CAT seemsto follow closely the same mode. However,the amount of DAS detectable in derepressed cells is relatively low compared to the methanol-induced status.

3 CLONINGANDCHARACTERIZATION OF METHANOL METABOLISM GENES Several genes encoding enzymes of the methanol utilization pathway of the two methylotrophic yeasts have been cloned and characterized. Besides two unidentified open reading frames encoding proteins of 76 and 40 kDa, respectively, a gene encoding an alcohol oxidase (AOX1) was isolated from P. pastoris in 1985 (27). Two years later, a second highly homologous gene(AOX2) was discovered (14). Both genes contain a1992 nucleotide open reading frame encoding enzymes of some 74 kDa (77). A structural comparison revealed two coding sequences92% of and 97% homology at the nucleotide and the deduced amino acid sequence levels, respectively (77). Codon usage exhibited a strong biasto triplets similar to moderately and highly expressed S. cerevisiae genes (78). In contrast to this high homology found within the coding region, no longer sequences of homology were observed in either the 5' or 3' noncoding region. The

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primary transcription site was found 115 nucleotides upstream in both genes. Transcription startswith an adenine which is surrounded by a pyrimidine track. The putative RNA polymerase binding sites (TATA box) are in both cases at position -43 relativeto the primary transcriptioninitiation site. The DNA sequences surrounding the translation initiation

site(CGAAACGATGGCTinAOXl,GAGAAAAGATGGCCinAOX2) were found to follow closely a constrained consensus sequence( A A X X A A A ATG GYT) deduced from highly expressed genes of S. cerevisiae (7981). Within the 3' region of both genes, sequences are present exhibiting to function in3' mRNA some similarities with consensus sequences thought processing, as suggested by various authors (78-80). In H. polyrnorpha, a single methanol oxidase (MOX) gene has been isolated and characterized (23).The gene harbors an open reading frame of 1995 nucleotides encoding a polypeptide of calculated 74,050 Da. The amino acid sequence deduced from this gene exhibits a homology of more than 85% with the Pichia counterparts. However, the codon usage differs greatly in favoringG/C containing triplets,thus following the high G/C content of the H. poljmorpha genome (24).The 5' untranslated regionof mRNA is veryshort and constitutes some20 to 25 nucleotides, as shown -50. The transby S1 mapping. The TATA box is located around position lation initiation sequence ATCA TG AXG exhibits some deviation from the consensus sequence described above. In addition to MOX, the single-copy genesfor formate dehydrogenase ( F M . )(25), dihydroxyacetone synthase(DAS) (M), and catalase (CA7) (26) have been isolated. TheFMD gene harbors an open reading frame of 1085 nucleotides encodinga 35,700-Da protein, DAS a 2109 nucleotide open reading frame for a potential 77,000-Da protein, and CAT a 1521 nucleotide open reading frame encoding a 507-amino acid enzyme. All genes are characterized by the codon usage bias for G/C-containing triplets as reported for MOX. Again, the 5' untranslated regions of the deduced mRNAs are very short, consisting of 15 to 25 nucleotides. Comparative sequence analysis of genes encoding peroxisomal proteins and the analysis of N-terminal amino acidsof the mature protein did not reveal N-terminal leader structures within the deduced amino acid sequence as observed for proteins sortinginto mitochondria orinto the secretory pathway (84,85). However, studies on localization of gene products derived from mutagenized or trucated genes indicated that import into the organelle requires specific targeting signals within the primary sequence of the polypeptide. These signalswere shown to reside in the C-terminus of the protein. A C-terminalS-K-L tripeptide was found to be necessary

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and sufficientfor an organelle import in mammalian cells(86-88). From sequence comparisonof several genesfor peroxisomal proteins a consensus sequenceS-, A-, C-/K-, H-, R/L was deduced (87,88).The targeting signals for the peroxisomal proteins of methylotrophic yeasts reside in the C-terminus aswell. They all show deviations from the S-K-L motif present in mammalian proteins and from the proposed consensus sequence described above. The two alcohol oxidases from Pichia and the methanol oxidase from Hansenula contain a C-terminal A-R-F motif (23,77), the dihydroxyacetone synthaseN-K-L (U), and the catalaseS-K-I (26). This suggests that the targeting motifis less conserved than originally reported (91,92). Furthermore, peroxisomal thiolase was recently shown to contain targeting signals within a cleavable N-terminal leader, reported for the first time for peroxisomal-imported proteins (89,90). The 3’ end regionsof the variousH. pofymorpha genes analyzedso far contain some segments that exhibit a weak similarity characteristic for transcriptional termination and polyadenylation signal sequences. The DAS 3’ region harbors sequences with somestructural similarities to that of the S, cerevisiae ADHl gene (82); the FMD and MOX gene share a TGAA...(A)..ATAGG motif that is repeated three and two times, respectively(93).Analyticalapproachesquantifying transcripts showed that the different methanol pathway genes are controlled primarilyat the transcriptional level (57,58). Detailed studies with MOX promoter variants fused to a bacterial P-lactamasereporter gene revealedthat this region is composed of several positively and negatively cis-acting regions (94,95). A 1.5-kb segment was found to be necessary for full promoter strength. Withgel retardation assays and DNase Ifootprinting, two activating regions have been identified which consist of binding sitesfor yet unknown proteins (94,96). A first element UAS1, defined by footprint and deletion analysis, covers a region between -484 and -471 relative to the ATG start codon of the gene and itwas appointed to a 14-nucleotide 5TCCTTGCACCGCAA3’ motif. Deletion of this region results in20-a fold reductionof reporter gene expression. It could be shown that the unknown factor binds to this element under repressed, derepressed, and induced conditions (95).A sequence highly homologousto this MOXelement with only a single T-to-C exchange is present inCATgene the from position -415 to -401 relative to the start codon.Since this type of element was that it might not detected either in DAS or in FMD the gene, it is assumed be the binding sitefor a protein involved in oxygen-dependent regulation (94). Sequence homologies place UASlinto close relationshipto the welldefined UAS2 of the S. cerevisiae CYCl promoter (95). A second element

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UAS2 was located at position -732 to -718 relative to the MOXstart codon and consists of the sequence5' TCGAGGTCGTGG 3'. This element has no homologyto the first described above and thus probably corresponds to a different regulatory signal. third A element between-921 and -892 is characterized by deletion analysis as an upstream repressing sequence. (95). Since all fourH. polymorpha genes exhibit some degree of coordinated regulation (see the preceding section), a searchfor other sequences with structural similarities as potential regulatory segments within the promoter region of these geneswas tempting. Indeed, a remarkable stretch of homology has been identified in a region about l o o 0 nucleotides upstream of the translation initiation codon. A consecutive segmentof 65 nucleotides of theMOX promoter shows high homologyto a 139-bp region inthe DAS regulon, interspersed by several nonhomologous sequences (93; see also Fig. 3). A similar segment of homology not is found in any other region ofboth genes. When the 0-lactamase reporter gene was fused to a shortened MOX promoter that did not contain this element, only weak expression was observed in methanol-induced cells transformed with this construct (57).

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TAGATATTITCTGCC * TCGTCGTACI'CA-54N-GTGTGATG8N-TCACC-9N-

TCGAAATlTT.TGCCGTCGTCGTAC**A GTGTGATG TCACC m-1052

ATCGCTTCGTACTCGCI'CI'GCAGCIT*CGA

-

ATCGMT GTA AT GAG CI'GCAGCITGCGA -987

Figure 3 Homologous sequences in the DAS and MOX promoters. Sequence comparison of the two methanol-inducible genes revealed an extended stretchof homology in a region about lo00 nucleotides upstream of the translation initiation codon in both genes. (From Ref. 93).

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Further stretches of homology can be deduced from sequence comparisons of the various promoter structures. However, no evidence has been presented so far to attribute a specific functionto these sequences. No sequence has been identified that is common for all methanol-inducible genes despite the fact that they all share a similar induction mode.

3.1 ChromosomalLocalization of MethanolUtilizationGenes In Hamenula, six chromosomes can be separated by pulsed field gel electrophoresis. FMD, MOX, and DAS sequences hybridyze to different chromosomes, I, 11, and VI, respectively, revealinga distinct localization of the methanol utilization pathway genes (97; unpublished results). 4 GENETIC ANALYSIS

The genetic analysis of methylotrophic yeasts is less developed than knowledge of their physiology. The most advanced species with respect to a comprehensive genetic analysis is Pichia pinus. This organism exhibitsa high frequency of sporulation, and the spores are characterized by a good vitality. A wealth of mutants exist and genetic maps couldbe established (98,99). LikeP.pinus, H. polymorpha is consideredto be a homothallic organism, although some evidenceof a bipolarity of mating types exists for both organisms (100,101). A system to perform genetic studies has been developed for both yeasts, but the information published so far is relatively sparse. However,the growing interest in these species has stimulated a rapid increase of knowledge during the past several years. The newly generated mutants for the analysis of peroxisome biogenesisand function described before attest to genetic approachesfor a more comprehensive understanding of these organisms (73-75). Life-cycle stages have been defined. Both organisms have a stable haploid phase under standard culture conditions. Aftertransfer into conditions of restrictive growth, fusion of cells and formation of zygotes occur to some extent. Under further restrictive conditions these zygotes enter meiosis and sporulate. However, after retransfer into a rich medium, the zygotes remain in a stable vegetative diploid phase, which subsequently can be induced under suitable restrictive conditionsto produce asci from which two to four haploid ascospores derive. After germination, haploid coloniesare generated again(101). It appeared to be difficultto obtain some specific types of auxotrophic mutants. Severalauthors observed a strong bias in the types of mutants selected. In H. polymorpha, no useful tryptophane

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pathway mutants could be recovered from a particular strain, whereas ura and ade mutants could be identified at a high frequency.As a possible explanation, the existence of mutationalhot spots and/or a possible aneuploidy has been suggested by various authors (104,105). Nevertheless, mutants suitable to serve as useful auxotrophic hosts for heterologous gene expression have been isolatedfor both organisms (102). The genera Candida and Torulopsis do not have a sexual phaseand thus it is not possible to develop classical genetics.

5 EXPRESSIONSYSTEMSBASEDON METHYLOTROPHIC YEASTS H. polymorpha and P. pastoris have both been developed as hosts for heterologous protein production (4,5). Auxotrophic hostsare transformed with DNAcontaining suitable complementing genes or dominant marker genes for selection. The foreign genes to be expressed are fused to the strong and tightly regulated promoter structures derived fromthe methanol utilizationgenes described before. Vectors can be designed for intracellular production orfor efficient secretion of the recombinant protein (see Section 6). In H. polymorpha, two auxotrophic mutants in either orotidine 5'-phosphate decarboxylase (ura3) (9) or in P-isopropylmalate dehydrogenase (leu2)(10) have been used mostly for transformation. Accordingly, vectors for transformation of these strains have been designed harboring the homologous or the correspondingS. cerevisiae genes for selection (9,lO). In Fig. 4 a representative exampleof an H. polymorpha expression vector designed for transformation of the uracil-deficient strain LR9 is documented (9). In addition to the selection marker, the vector contains further components. Thebla gene and the ori sequence are genetic elements for selection andpropagation in E. coli, and a HARS (Hansenulaautonomously replicating sequence) is a genetic structure for propagation in the yeast host. The presence of this HARS sequence is essential for an efficient transformation frequency of the host cells. However,transformation is possible with plasmide not containing HARS, generating integrants at a low frequency. The expression cassette consists of the tightly regulated FMD (in other constructs, MOX) promoter sequence, which provides a strong and stringently controlled expression. For termination of transcription, a terminator sequence derived from the MOX gene is present. Terminatorand promoter sequencesare separated by a multiple

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Asp718 coR1

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Figure 4 Structure of a H. polymorpha expression vector. For heterologous gene expression,the vector contains an expression cassette with a FMD promoter (FMD-P.) and a MOX terminator (MOX-T.) segment separated by a multiple cloning site for sequence insertion. In addition, the plasmid harbors the following components: the pBR322-derived bla gene and ori sequence for propagation in E. coli. For propagation in the yeast host, it contains a Hansenula autonomously replicating sequence(HAW)for selection ofthe H. polyrnorpha LEU2 and URA3 genes. The plasmid is used to transform leucing- or uracil-deficient hosts. In other constructs, the MOX promoter is used as a control element withinthe expression cassette. Modified vectors contain only a single complementation sequence or harbor a kanamycin resistance gene as a dominant selection marker.

cloning site for insertion of the heterologous geneto be expressed. Variants of this general design are available containing the LEU2 gene or both the URA3 and LEU2 genes for complementation, or a S. cerevisiae ADHl promoter/G418 resistance fusionas a dominant selection marker (103). A similar strategy was applied for the P. pastoris expression system (his4)was generated (13). A mutant deficient in histidinol dehydrogenase from a suitable parental strain. Plasmids containing the homologous HIS4

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gene or that from S. cerevisiae were designedfor transformation to select against the auxotrophic background. Again, the plasmid contains the pBR322-derived components for selection and propagation in E. coli. The expression cassette is composed ofthe AOX1 promoterand AOXl terminator sequences separated byan EcoRI site for insertion ofa sequence of choice. Furthermore, the commonly used plasmids contain an additional copy of the 3'AOXl terminator region positioned distant from the expression cassette. Thisis required for homologous integrationinto the AOXl locus of the P. pastoris host cell (Fig. 5). Transformation of both species follows versions of standard protocols that either require spheroplast formation (104) or employ polyethylene

Flgure 5 Structure of a P. pustorisexpression vector. For expression, the vector contains an AOXl promoter (5'AOXl) and terminator (AOXl-t) separated by an EcoRI cloning site for insertion of a heterologous sequence.The pBR322-derived components are as describedfor the Hamenula expression vector inFig. 4. For selection in a histidine auxotrophic host, the plasmid harbors the P. pastoris HIS4 gene. In addition, the vector contains a second segment of the 3' noncoding region of AOXl (3'AOX1), distant from thatwithin the expression cassette. The plasmid is linearized at the boundary of this segment (BglII digest) for sitedirected integration into the AOXl locus of the host. (Modified after Ref. 145.)

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glycol or Li salt treatment (105), or freeze-thawing methods which do not require protoplast formation (106). In our procedures we stick to a modified versionof the latter which enables long-term storage of competent host cells at -70°C (107). When the expression systems were established, integrative (vectors without the autonomous replicating sequences) and presumptive nonintegrative vectors (vectors harboring an ARS-like element) were used. In the first case, the heterologous DNA integrated readily into the genome. The fate of plasmids containing an ARS-like sequence (108) differed dependingon othersequences present on the plasP.pastoris-derived mid and on the type of ARS-like sequence used. When M S sequences (PARS1and PARS2) were present on vectors, the transformation frequency increasedand the plasmids were maintained as autonomous elements in recombinant P. pastoris cells at least for an exS. cerevisiae-derived ARS tended period of time (13). Using plasmids with sequences or whole S. cerevisiae 2-pm DNA instead, integrationinto the host’s genome was observed, indicating that the 2-pmDNA does not function as ori in P.pastoris (13). Similarly, PARS sequences did not function in transformedS. cerevkiae hosts. Since the Pichiu-specific PARS and the ARS sequences share sequence homologies, it has been argued that the autonomous replication functionis not restricted to this consensus region but might require additional species-specific genetic elements (13). In H. polymorpha, plasmids containing either a homologous HARS or an ARS sequence exhibit a high frequency of integration. It is still not very clear how long plasmids containing such elements are maintained in an episomal state before they eventually integrate into the genome. Tihomorova observed spontaneous formation of multimeric structures a plasmid encompassingan “imin recombinant strains transformed with perfect HARS element”,but this element evoked some kind of “autonomous behavior” (L. Tihomorova, personal communication).In our standard proceduresfor the generationof recombinant H. polymorpha strains containing integrated DNA, cells transformed with HARScontaining (40-60 genvectors first undergo several passages in a selective medium erations) and a subsequent stabilization on a nonselective medium. The resulting strains bear the heterologous DNA integratedinto the genome, and no free plasmid canbe detected sincethe episomal DNA is selected out during the growth on nonselective media. Strains can be identified that contain up to 100 copies of the foreign DNA, exhibiting a head-totail arrangement. In the example presented in Fig. 6, single-copy integrants and recombinants with up to eight copies have been identified. A

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a b c d e f g h i k l

094 kb 083 kb-

Figure 6 Copy number determination of integrated heterologous DNA in H. polymorpha. The genomic DNA of an untransformed host strain and different recombinant isolates of a particular construct were digested with the restriction enzyme XhoI. The restrictedDNA was separated on 0.8% agarose gels, transferred to nitrocellulose and hybridized to a 32P-labeled fragment of the cloned FMD promoter sequence. The hybridization pattern reveals two signals in similar electrophoretic positions, one for the genuine single-copyFMD gene, the other for the slightly larger heterologous fusion.In dilutions of the various DNA samples, the signal intensity of the foreign DNA can be compared to the intrinsic singlecopy control. In the example presented in this figure, recombinant strains with one to eight copies of the integrated DNA have been identified. Left lane, size marker; lane 1, untransformed host strain; lanes a to k, different recombinant isolates. In the arrow-marked lane (b) a four-copy integrant could be identified; in (c), a twocopy integrant; and in (h), an eight-copy integrant.

gene dosage effect of gene expression was clearly apparent in recombinant H. polyrnorpha strains. For instance, product yields obtained from a hirudin-secreting strain with 20 copies werefound to be much higherthan those from strains harboring 5 and 10 copies of the expression cassette (12,109). In glucoamlyase-secretingstrains, however, maximal yields of 1.4 g/l were secreted from a four-copy strain and no further increase in productivity couldbe obtained by strains with higher copy numbers. The reason for this exceptionally weak gene dosage effect in this particular case is unknown (1 10). The vectors commonly used to generate recombinantP.pastoris strains are integrative plasmids. They contain a further 3’140x1 segmentin

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addition to that within the expression cassette. The plasmids are linearized at the boundary of this sequence, targetingthe linearized plasmidto the host’s AOXl gene via homologous recombination (see Fig. 4). In other cases, the heterologous DNA is targeted to the P. pastoris HIS4 gene (1 11). HIS4 integrants were found to be the less stable type of HIS4-selected transformants, since recombination can result in the excision of the expression cassette, leaving behind theHIS4 gene. In contrast to the situation in Hansenula, homologous recombination usually results in the integration of a single copy of the heterologous DNA. To increase the yield ofa productthat appears to be gene dose-dependent in this species(1 11,112),protocols have been worked out to obtain multicopy integrants similarto those in recombinantH. polymorpha strains. They are obtained either using vectors containing multiple expression cassettes or by screening for multiple integration events (see also the next section). In both species, Hansenulaand Pichia, the transformants bearing the multicopy integrantswere found to be mitotically stable. Stable multimeric integrants have been identified for a P. pastoris strain expressing Bordatella pertussis pertactin (1 13) or a tetanus toxin fragment C producing strain (110),which will be discussedin more detail inthe next H. polymorpha strains producing different section. Several recombinant secretory or intracellularly expressed proteins were tested for mitotic stability by isolating and analyzing the heterologous DNA before and after growth for an extended period of time. Tests after 40, 100,and 800 generations confirmed the high stability. Molecular analysis revealing the genetic stabilityof a particularH. polymorpha strain expressing hepatitis S antigen (HBsAg) is documented in Fig. 7. In P.pastoris, the modeof integration might influence the level of foreign gene expression.In particular, this has been shown for strains with disrupted genomicAOXl generated by the transplacement event during transformation. During induction theseaoxl transformants do not simultaneously produce high levelsof the AOX enzymeand the heterologous product. They are characterizedby a slower growthrate exhibiting a much lower 0, demand comparedto wild-type strains. In a comparative study, expression of the Bordatella pertussis pertactin was analyzed usingstrains of slow growth (methanol utilization slow; MutS)and wild-type strains (Mut+)as hosts. The MutSintegrant expressed the recombinant protein Mut+ strain at 10% of total cell protein in a fermenter cultivation, while the reached onlya 5% level (1 IO). Fermentation protocols have been worked types of recombinant out to match the specific demands of these two basic P. pastoris strains (see Section7).

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Figure 7 Mitotic stability of aH. polymorphs recombinant strain H415 expressing HBsAg during growth on a nonselective medium. The medium (YEP) was supplemented with glycerolas carbon source. Rounds of inoculations into new media were carried out with cells transferred from late log-phase cultures. Twelve individual colonieswere isolated beforeand after a 40-generation growth period.The respective DNA was isolated and characterized by Southern analysis. Sufi/SspIdigested samples were hydrized to a 32Plabeled probe derived from the plasmid used for transformation. The hydribization pattern confirms that the foreign DNA is integrated into the genome and reveals its stabilityafter growth. Left lane, undigested DNAfrom strain H415;I, restricted DNA isolated from colonies before growth and 11. after growth; pRBS 269, plasmid DNA used for transformation (from Ref. 114) as control.

6 RECOMBINANTPROTEINSEXPRESSED IN METHYLOTROPHIC YEASTS Both systems have been applied successfully for the productionof heterologous proteins duringthe recent past and are used commercially in ongoing developments. An increasing number of intracellularly produced and secreted proteins attest to the quality of methylotrophic hosts for a summary is preheterologous gene expression. In the following section sented focusing ona few selected. representative product examples obtained

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from the two species;further aspects and examples are discussed in more detail in the forthcoming sections. 6.1 RecombinantProteinsIntracellularlyExpressed in Methylotrophic Hosts The possibility of a stable multimeric integration makes H. polymorpha an ideal host for the expression of complex composite structures suchas polyvalent vaccines. This was demonstrated convincingly by recombinant P.polymorpha strains coexpressing and L S hepatitis B envelope proteins in a balanced predeterminedratio, thus mimicking the compositionof the natural viral coat (1 14,115). The envelope of the hepatitisB virus (HBV) is composed of the S-antigen as the major viral protein and two minor related proteins, the M- and L-antigens (116). S-antigen was expressed successfully inS. cerevisiae inthe early 1980s (117). Vaccines formulated with S. cerevisiae-derived hepatitisB surface antigens have been available commercially for several years(1 18,119). The recombinant vaccines consist of the S-antigen alone, which is assembled in vivo in the yeast host into highly immunogenic 22-nm particles similar to those found in the serum of hepatitis B patients. S-antigen-expressing strains have also been obtained for P.pastoris (120) and H . polymorpha (114,115) which were found to produce up to 0.4 and 0.5 to 0.6 g/L, respectively. Since additional protective epitopes are present in the L-protein which are absent in presently available recombinant vaccines (121), strains were constructed simultaneously expressing both theS- and L-antigens(114,115). The composite particles derived from such strains might provide the base for an improved vaccineor diagnostic probe. To construct such strains, the uracil-deficient H. polymorpha hostLR9 was transformed with a plasmid harboringan expression cassettefor the 3. Mitotically stable L-antigen analogousto the example presented in Fig. strains were obtained containing one or several copies integrated into the genome. Selected recombinant strains characterized by different L-antigen expression levels were transformed again with an S-antigen expression vector. This vector contained as a dominant selection marker the Tn5derived kanamycin resistance gene expressed from S.thecerevisiaeADHl promoter (130). Different strains were identified as producingboth proteins, with fixed L/S ratios ranging from 1 :8 to 1 :15, which correlates with the dose of integrated genes. The proteins producedwere assembled into particles, now containing both antigens in the ratios noted above (Fig. 8). During fermentation, the ratio remained constant due to the stable

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Figure 8 Analysisofrecombinant HBsAg particles (small hepatitis B virus surfaceantigen)producedin H. polymorpha. (A)Quantification of theexpressed antigens by Western blot analysis. Different recombinant strains were constructed with either a FMD- or a MOX-promoter controlled expression. Crude extracts from the various isolates were assayed from HBsAg content with specificmonoclonal antibodies comparing the immunological signal of the different samples with those from purified HBsAgstandards. Lanes 1 to 5, HBSAg standards containing 0.03, 0.07, 0.15, 0.3, and 0.6 pg of protein, respectively; lanes to 6 to 8, 3-, 6-, and 12-pg samples (total soluble protein) from strain H415 crude extracts (MOX-promotercontrolled expression of HBsAg); lanes9 to 11, 3-, 6 ,and 12-pg samples from strain H352 crude extracts (FMD-promoter-controlledexpression of HBsAg). (B)Identification of HBsAg particles by sucrose gradient centrifugation. Crude extract from strain H415 was centrifuged in a linearto20 50% sucrose gradient. After centrifugation the gradient fractions were assayed for antigen content by Western blot analysisas described above and by a particle-specific Auszyme assay. The peak indicatesthe efficient formation of 22-nm particles. (C)Electron micrograph of 22-nm HBsAg particles. HBsAg particles preparedfrom H. polymorpha strain H415 crude extract were depositedon a colloidon gridand stained with uranyl acetate. Magnification x 180,000. (From Reg. 114.)

integration of the heterologous genes within the host’s genome and the well-balanced functional promoters in both expression cassettes (1 14, 115). A potential subunit vaccine against tetanus, the tetanus toxin fragment C, was produced intracellularly inP.pastoris (1 11). Tetanus fragmentC is a 50-kDa polypeptide derived by papain cleavage from Clostridium the

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tetani toxin (122). The development of a recombinant production system might providea safe nontoxic alternative production process. The current vaccine consistsof formaldehyde-inactivated tetanus toxin, which has to be produced in a potentially hazardous process(123). For expression in P.pastoris, a plasmid was designed analogous to the example shown in Fig. 4. A synthetic sequence encoding the fragment C was insertedinto the cloning site ofthe expression cassette.The resulting vectorwas linearized for targeting into specific loci of the host’s genome. Using different strategies, plasmids were either targeted to transplace theAOXl or targeted to other chromosomal loci. By the first procedurethe genuine gene is disrupted and the original gene function is lost, resulting in slow methanol utilizing mutants (MutS). Alternatively,the DNA was linearized in such a way that the intactness of the AOXl function was not affected by the crossover event, leading to recombinant strains of the Mut+ type. In a third approach, the foreign gene was targeted to the HIS4 locus of the host (see also Section5). The expression of the proteinwas found to be independent of the site and mode of integration, contrastingthe finding for recombinant pertactin-producing strains described before. As in the H. polymorpha example above,an increase in the expression level correlates with the gene dosage. Multicopy integrants canbe obtained either by constructing vectors with several expression cassettesbyorscreening for multicopy integrants. They could be distinguished from transformations using DNA fragments targeted for single-copy transplacement. It is assumed that they arose by circularization of the transforming DNA followed by multiple integration into the chromosome via repeated singlecrossover recombination events(1 11). In scale-up experiments, yields of several grams per literof biologically active material could be obtained.

6.2 Recombinant Proteins Secreted from Methylotrophic Hosts Eukaryotic secretory proteinsare targeted to the secretory apparatus by N-terminal leader sequencesthat are removed from a precursor protein on entry into the endoplasmic retriculum(ER). For many proteins, passage through the secretory pathway is a prerequisite for proper folding by formation of S-S bridges and appropiate protein modifications such as glycosylation. For secretion of a recombinant protein, it is necessary to fuse the polypeptide to an N-terminal leader sequence that directs it to the secretory apparatus of the host cell. A variety of different leader sequences has been usedto drive secretion of heterologous proteins in yeasts. The most common elements in the traditional S. cerevisiae host are derived

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from genuine genes encoding secretory proteins: that for invertase (SUC2) (124), acid phosphatase (PH05)(125), and the a mating factor (MFal) (126). Leader sequences usually consist of some 20 amino acids and are composed of a hydrophobic core 6ofto 15 amino acids framed by hydrophilic amino acid residues. A theoretical model deduced from a compilation of protein sequences allows prediction of the processing(127). site In the case ofMFal, a two-step maturationof a prepro-polypeptide takes on entry into the ER (128) as stated place. First,a leader sequence is cleaved above. The remaining propeptideis further processed by the proteolytic activity of the KEX2 protease within the Golgi apparatus (129). In most constructs using this leader, the heterologous sequence is fused to this KEX2 recognition site. In the two methylotrophic hosts, fusions with these leader sequences and those from other eukaryotic sourceswere found to be faithfully processed, indicating a compatible mode of secretion and of cleavage atthe Lys-Arg ( K E X 2 ) processing. As a single exception, lack processing siteand inappropiate N-terminal extensions ofthe mature secreted protein have been reported for a prepro-MFdaprotinin fusion expressed in a P. pastoris host. It is assumed that structural properties of the aprotinin force the inaccurate removal of the preprosequence(130). In glycoproteins, oligosaccharidesare either N-linkedto an asparagine residue or 0-linked to a threonine or serine residue of the polypeptide chain. In N-glycosylation,a core consisting of nine mannose, two N-acetylglucosamine, and three glucose residuesis added to an asparagine within the ER. After subsequent removal of a mannose andthe glucose residues, further processing take place within the Golgi apparatus, resulting indifferent typesof oligosaccharide side chains. S. cerevisiae lacks Golgi mannosidases, by whichfurther mannose residues are removed in many other eukaryotic cells. This removal is necessaryto generate high-mannose or complex-type side chains typically found in animal cells. Instead, further mannose are added, resulting in heterogeneous side chains with up to 75 mannose residues (131). As a consequence, many heterologous proteins are overglycosylated (>40 mannose residues within a side chain) when secreted from a S. cerevisiae host (132). In methylotrophs, overglycosylation was not observed in many secreted recombinant proteins (see below). As exceptions, overglycosylation is reportedfor a recombinant aglactosidase secretedfrom a H. polymorpha host (133) and HIV gp 120 secreted from a P.pastoris host (C.A. Scorer, cited in Ref. 8). In the first example describing the production aofsecretory protein in H. polymorpha, the glucoamylase(GAM) gene from the amylolytic yeast Schwanniomyces occidentah (134) was expressed usingthe genuine leader

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for export. Forthis purpose a plasmid was constructed by inserting a fragment withthe entire coding region, including the leader sequenceinto the H. polymorpha expression vector. The heterologous gene was expressed from a FMD promoter. Again, transformation with the final construct resulted in several recombinant strains harboring different copy numbers of the expression cassette stably integrated into the genome. Strains with one to eight copies couldbe identified. A particular strain with four copies was analyzed in more detail.It was found to secrete 1.4g of biologically active enzymeat a cell mass of130 g dry weight per liter (see also Section 7). When the recombinant enzymewas compared with its S. occidentalis counterpart, identical N-termini were determined demonstrating a correct processing ofthe signal sequence. Comparative analysis of the sugar moieties revealedan identical extent ofN- and O-glycosylation of the two polypeptides, as shown in Fig.9 (1 10). No overglycosylationW& observed for invertase (see below) and L-antigens from the hepatitisB virus which are typically overglycosylatedin S. cerevisiae. Obviously, the expressed Lantigens enter the secretory pathwayto some extent. High-level secretion of 2.5 g/L was observed in recombinant P.pas(135). The invertase expression vector was toris strains secreting invertase constructed by ligating an S. cerevisiae invertase (SUC2) gene (124) sequence into the cloning site of the expression vector. The sequencewas composed of a suitable gene fragement and a synthetic oligonucleotide encompassing the N-terminal leader sequence designed accordingto the genuine gene. The resulting vector was targeted for transplacement into a Suc+ phenotype. the AOXl locus. The resulting transformants exhibited Again, an appropiate signal peptide cleavage was observed. Comparative electrophoretic analysis revealed differences between the S. cerevisiaederived enzymeand the recombinant product. Invertase from S. cerevisiae exhibited a highly variable ektent of glycosylation, yielding proteins ranging in size between1 0 0 and 1 4 0 kDa (136). In contrast, the P.pastoris-derived product appeared to be a more homogeneous protein with a molecular weight of some85 kDa. Deglycosylation ofboth species in both cases resulted in proteins with a molecular weight58ofkDa. A detailed analysis of the size distribution and the general structural features of the sugar moieties revealed that the invertase produced inPichia contained more than 85% of the N-linked oligosaccharides as structures in the size range of Maq,,GlcNAc+ Thus glycosylation of the recombinant invertase resembles closely the pattern observed in high-mannose oligosaccharides of glycoproteins in higher eukaryotes (137). When invertase was expressed in a Hansenufa host, a comparable type of glycosylation was observed (138).

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Figure 9 Glycosylation of recombinant glycoamylase expressed in H. polymorph. Glucoamylase (GAM) was isolated from the culture medium ofa recombinant H. polymorpha strain expressing the GAM gene from Schwanniomyces occidentalis. The isolated protein was deglycosylated withEndo H. Samples of the undigested and the deglycosylated enzyme wereseparated through 7.5% gels and visualized by staining with periodic acid-Schiff reagent for glycopeptide detection (153). The electrophortic mobility ofthe proteins was compared to thatof isolates from S. occidentalis. An identical extent of N-and O-glycosylation was found for both enzymes. Lane a, undigested heterologous GAM; lane b, deglycosylated recombinant GAM; lane c,deglycosylated GAM from S. occidentaliq lane d, undigested GAM from S. occidentalis. (from Ref. 110.)

Correct foldingand formation of S-S bridges in the appropriate position was clearly demonstrated for heterologous products secreted from both hosts. Examplesare recombinant aprotinin secreted from a P. pastoris host (130) or hirudin and miniproinsulin synthesized by recombinant H. polymorpha strains (U. Weydemann, Rhein Biotech GmbH, personal communication). Many proteins have been produced at high levels in the two methylotrophs. These include the foregoing examples of intracellular expression and the described secretory proteins. When the yields of specific heterologous proteins produced in different expression systems were compared, methylotrophs werefound to be at least as efficient as other yeasts or proved to be the superior hosts (6). The increasing number of

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heterologous compounds expressed in the two methylotrophic yeasts comprise a variety of proteins, including enzymes, hormones, anticlotting factors, and vaccines ranging in size from 7 to 150 kDa. A compilation of the different polypeptides produced the in two species is given in Table 1.

7 FERMENTATION An advantage of the exploitationof the two methylotrophsfor heterologous protein productionis the possibility to combine the control of gene Table 1 Production of Foreign Proteins in MethvlotroDhic Yeasts

Yeast

Protein

Promoter

Refs.

H. polymorpha

P-Lactamase" HBsAg Pre-S2-HBsAga Pre-SlS2-HBsAga cr-Galactosidaseb Glucoamylaseb Hirudinb Human serum albuminb Invertaseb Mini-proinsulinb

MOX FMD MOX FMD MOX MOX FMD MOX FMD FMD MOX MOX FMD

P.pastoris

P-Balactosidase HBsAg Tetanus toxin fragment C Pertactina

AOX AOX AOX AOX AOX AOX AOX AOX

138 114,115 153 114,115 132,152 110 12 151 138 U. Weydemann, personal communication 146 120 111 133 147 148 149 C. A. Scorer, cited in Ref. 8 135 143 112 150 130 Cited in Ref. 8

TNFe Streptokinase

sow

HIV g p 1208 Invertaseb Bovine lysozymeb Murine EGF6 Human EGI? Aprotininb Human serum albuminb aIntracellular expression. bSecretory Proteins.

AOX AOX AOX AOX AOX AOX

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expression by strong and stringent promoter elements with the favorable fermentation properties of these yeast strains at an industrialscale. These yeasts can growto extremely high cell densities(130 to 150 g dryweight per liter culture medium), excel by fast growth rates, and feed on cheap carbon sources such as glycerol and methanol. The useof glycerol is advantageous for a fermentation process since it results in a very limited level of ethanol formation. Ethanol negatively influences the production process of heterologous compounds in scale-up fermentations. This is found to be a problem in recombinantS. cerevisiae strains, which cannot use glycerol efficientlyand which often produce higher levels ofethanol in glucose-supplemented media(139). The commonly used culture media for methylotrophs (Table2) are based on simple synthetic components. They contain trace metal ions and adequate nitrogen sources which are required for efficient expression and cell yield (140,141) and do not contain proteins. Since thesecell strains normally do not secrete many proteins that may contaminate or even degrade heterologous products, they can easily be purified. High stability and lackof degradation was shownfor several proteins secreted fromH.a polymorpha host when stored in culture supernatants for a long period of time. In addition, no other major proteins or substances interfering Table 2 StandardMedium for H. polymorph# m4)ZS04

G&)PO, CaCl, MgSO, 7&0 NaCl FeCI, 6&0 CuSO, 5&0

ZnS04-7H,0 MnSO,.H,O Ni SO, 6H,O CoCb 6H,O

K1 %BO, mg/L N%MoO, d-Biotin Thiamine HCl

-

3.45 g/L 1.33 g/L 0.03 g/L 0.24 g/L 0.04 g/L 1.2 mg/L 1.2 mg/L 4.05 mg/L 0.5 mg/L 0.15 mg/L 0.15 mg/L 0.15 mg/L 0.15 0.15 mg/L 0.02 mg/L 2.7 mg/L

%Suitableamounts of a carbon source haveto be added.

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(12,110).

The stable integration of the foreign DNA adds further to the superior fermentation characteristics of the two yeasts. The production process is not impaired by plasmid instability or plasmid rearrangements observed in transformants with episomal heterologous DNA (142). Any nonselective medium can be used, and a medium can be designed or optimized to match the special requirements and demands of an economic production and downstream process fora product of choice. In the case of recombinant H. polymorpha strains, different fermentation modes can be distinguished. The mode selection depends on the promoter sequence used for heterologous gene expressionand the inherent control mechanism imposed by this structure (see previous sections)and may vary with the type of proteinto be expressed. Ina twocarbon-source mode, the recombinantstrain is first grown on high glycerol or glucose concentrations to a certain cell density, in some casesup to 80 to 100 g dry weight perliter. At the endof this growth phase, as the concentration of the first carbon source is depleted, methanolis added as1 to 1.5% (v/v) to the medium. Fifteento twenty hoursafter methanol addition, a strong induction of the methanol-inducible promoter is observed resulting in a massive expression of the heterologous compound. Maximal productivity is reached after 20 to 30 h. The total fermentation time for both phases depends on the strain, the culture medium,and on propertiesof the protein produced and varies between 70 and 120 h. This two-phase fermentation mode assures a high per cell productivity to duean optimal induction of the heterologous expression cassette. Both promoters,the MOX and F M . promoters, respectively, can serve as control elements and proas a repressive carbon source vide an efficient expression. The use of glucose during the first step of fermentation offers the possibility to express compounds that might interfere withcell growth orthat are potentially hazardous or toxic to the cell. The disadvantageof this two-step mode isthe necessity to change the carbon source during the production process. This requires careful controlof the carbon compound levels inthe culturemedium, but a highly reproducible process can be established. A possible mode of fermentation would beto perform the entirefermentation process on methanol. However, since the growth rate with methanol is low, it is advisableto grow the cells initially on another carbon source and to switch later to methanol for the production phase as described above. The second fermentation mode is based on the high activity level of the FMD promoter under derepressed growth conditions. In this process, glycerol is used as a sole energyand carbon source. This typeof

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fermentation startswith a feed of 1to 2% glycerol, resulting in rapid growth of the culture to high cell densities. Upon almost complete depletion of glycerol at an anticipated cell mass level, glycerol is maintainedat concentrations between 0.1 and 0.3% (w/v). Under these conditions both promoters are derepressed (see Section2). The advantage of this mode is the easycontrol of the process,and for some applications glycerol-grown cells were found to perform better than those grown on methanol. For instance, the cellsare bigger and the cell wallsare less rigid. Although the per cell productivityis 20 to 50% less than that observed inthe two-step fermentation mode, the economy of the process can be superior due to a shortened fermentation time and the possibility of reaching very high cell densities. This mode is especially advantageous in a FMD-promoterdriven production process. The activity of the FMD promoter reaches50 to 90% of the induced status under these conditions. maximal yields were found to be correlated with cell mass over a wide range when glucoamylase was expressed from a FMD promoter (110; see Fig. 10).

0.75

dry weight ( m

Figure 10 Correlationbetweenmaximalyields of secretedglucoamylaseand cell mass. Cells were grown in 3% glycerol (w/v) to different densities before being transferred to induction conditions inmethol. Growth continuedfor at least 24 h before yield determination. Filled circles represent yields routinely obtained in fermentations of recombinant strain harboring four copies of the heterologous expression cassette. (from Ref.110.)

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As an example, a one-step fermentation of a hirudin-secreting H. polymorpha strain is documented in Fig. 11 exhibiting some of the features stressed before. For fermentation studies, a recombinant strain was constructed harboring 20 copies of an expression cassette, stably integrated into the chromosome (12). Secretion was achieved by fusing the hirudin sequence to the S. cerevisiae MFal preprosequence. A productivity of to 110g dry weight. The HPCL 1.8 g/L was observedat a cell density of 80 analysis indicated perfect folding and proper processing of the signal sequence. Recombinant hirudinis especially susceptibleto proteolytic activity, by which the C-terminal amino acidsare removed to yield a shortened protein. This shortened form is typically recovered in high amounts when S. cerevisiae is used as an expression system. The highamount of Hansenula is due intact hirudin (more than 80% of the expressed protein) in to some extent to intrinsic properties of the methylotrophic host, but it was further improvedby a suitable medium composition (data not shown). In a modified version the of one-step fermentation mode, a feed becan initiated usinga mixture of glycerol and methanol as a carbonsource during the entire fermentation process. This type of process results in a superior per cell productivity, but the fermentation time is usually longer. Both promoters, MOXand FMD perform perfectlywell under these conditions. In Pichia pastoris, the control mechanism of the methanol metabolism pathway genes differs,as pointed out earlier. The AOXl promoter, which is homologous to the MOX promoter in Hansenula, is the only component commonly used for control of heterologous gene expression. Since strong derepression is not observed, fermentation and scale-up processes are established which closely resemble the two-step fermentation mode described for H. polymorpha. A convincing example is represented by recombinant P. pastoris strains expressing and efficiently assembling the

Figure 11 Fermentation of H. pobmorpha strain H-22 secreting a hirudin (F” promoter controlled expression). Strain H-22 was grown in a batch-fed 10-L fermentor on a semisynthetic nonselective medium supplemented with glycerol as carbon source. In the first growth phase, cells are cultured to high cell densities with a high-level supplementation of glycerol; the subsequent production phase is characterized by low levels of glycerol. The insert documentsHPLC analysis of the medium sampled at the end of fermentation. The undegraded 65-amino acid form of hirudin (peakH) constitutes some 80% of the secreted hirudin (back filled peaks inthe chromatogram) indicatinga low amount of products degraded at the C-terminus. For further details, see the text.

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hepatatis B surface antigen (HBsAg)(120). First the strains were grown in a defined minimal medium with glycerol as a carbon source. Upon depletion, methanol was added. Cells were harvested200 h after a shift to inductive conditions, and yields of 0.4 g/L were obtained at a cell density of 60 g dry weight per liter. This productivity was similarlyfound in scaleup fermentations at a 240-L scale when a similar mode of cultivation was applied (120). In another example, the fermentation conditions were optimized for recombinant P. pastoris strains expressing bovine lysozyme (143). The fermentation developmentwas carried out for strains of the MutS (disruption of the host’s AOXl gene by the recombination event)and the Mut+ (recombinants withan intact AOXl gene) type.A first approach employed a fermentation protocol comparable to that recombinant Muts strains. In described abovefor the HBsAg-producing strain, an induction phase required 175 h to yield maximal lysozyme levels of 250 mg/L at a cell density of60 g dryweight per liter. Foran attempt at improving productivity, the compositionof carbon compounds were altered duringthe induction phase. In a mixed feed of glycerol and methanol in a 4:l ratio (3.6g of glycerol per 0.9 g of MeOH per hour) a maximalproduct concentration of 180 mg/L was obtained within39 h at a cell density of82 g dryweight per liter. When the composition was altered to a 2:1 ratio, 290 mg/L was 85 g dry weight. In a modified version of yielded in43 h at a cell mass of the 2:l protocol, the MeOH feed was increased to a point where excess (nonlimiting) methanol beganto accumulate. This type of fermentation yielded an amount of 375 mg/L within 45 h at a cell density of 103 g dry weight per liter. Thus an up to fourfold increase in volumetric productivity (mg/L-h) over the original methanol feedwas observed, but the per cell yield (5.2 mg/g dry weight in the methanol feed) decreased, ranging from 4.0 mg/g dry weight (modified2:1 version) to 2.3 mg/g dry weight (4:1). In the second approach to shorten thetotal fermentation time, recombinant strainsof the Mut+ type were analyzed. Mut+ strains are sensitive to changes in the residual methanol level. Therefore, a three-step mode of fermentation was applied. First, the cultures were grown in glycerol excess. In a second step, a feed was initiated with lowamounts of glycerol. This provides afurther increase in cell massand allows that a weak derethe AOXl propression of the AOXl promoter (under these conditions, 2% of the induced status, as mentioned earlier moter activity reaches some in Section2). In a third phase, production is induced aby ramped methanol 450 mg of lysozyme per literat a feed. This fermentation protocol yielded

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cell mass of84 g/L. In scale-up fermentationsto a 10-Lvolume 560 mg/L was obtained within 30 h. Thus this type of optimization forced a 6.5fold increase in productivity relative to the original Mutsstrain. In this protocol, a smooth transition from the growth to the production phase is provided by the intermediate feed on limited glycerol concentrations. In this fermentation mode, a stringent control of medium components has to be obeyed. In contrast, this smooth transitionis readily obtained in Hansenula. Due to the high activity levels of methanol pathway genes under derepressed conditions,the simple one-step fermentation modeor modified versions of it can be applied as described before.

8 DIFFERENCES BETWEEN THE H. POLYMORPHA AND THE P. PASTORlS EXPRESSION SYSTEMS In the previous sections we documented the most promising performances of the two methylotrophic yeast speciesas hosts for heterologous gene expression. In both casesthe cells are shownto grow to very high densities (100 to 130 g dryweight per liter). Generally, these high densities are obtained within a short fermentation time(see Sections 5 and 7 ) , although some HBsAg-producing P. pastoris strains are characterized by a very long induction/production phase of 80 to 100 h (135). Integration of the heterologous sequences resulted in mitotically stable recombinant strains (see Section 5 andFig. 6).Both organisms followa similar control mechanism for the promoters of methanol utilization genes, which are exploited as control elements for the expressionof foreign genes (see Sections2, 3, and 5). Despite the obvious similarities, the two systems were shown to differ in the following aspects. Transformation inP.pastoris usually resultsin the integration of a single copy of the heterologous sequence. It is only recentlythat protocols have been workedout to obtain strains with multiple copies comparable to the situation in Hamenula, as described before for the tetanus toxin fragment C-producing strains. For expression in Pichia, foreign genes are fused to the AOXl promoter. In Hansenula, two strong methanol-inducible promoter structures are applied for heterologous gene expression: the MOX and FMD promoters. In Pichia, methanol is the only carbon compound that elicits high levels of methanol-inducible proteins. HanIn senula, expression of these proteins is also observed in glycerol-grown derepressed cells. This allows the elimination of a methanol induction step. FMD-promoter-driven production especially can be obtained in strains grown on a mixture of methanol and glycerol or on glycerol alone, en-

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abling a one-step fermentation and resulting in a high production rate. The differences betweenthe two systems are summarized in Table 3.

9 CONCLUDING REMARKS The capabilitiesof the methylotrophs presented in this chapterattest to the favorable characteristicsof these yeasts as expression systems for heterologous proteins. Their performance as hosts for recombinant protein production make them highly competitive in comparisonto other commonly used bacterial, yeast,and eukaryotic cell line systems.To a large extent they meetthe characteristics generally desiredfor an efficient industrial host, including rapid growth on low-cost media, stringent control of a reproducible production process, high expression levels ofthe desired product using both an intracellular and a secretory expression mode, proper folding, and appropriate modifications. Limitation may be encountered in cases where a complex mammalian type of N-linked glycosylation is indispensable.An appropriate glycosylationwas observed in several recombinant proteinsthat were found to be overglycosylated when secretedfrom a S. cerevisiae host. This indicatesthat the methylotrophs are able to express structures similarto the mammalian high-mannose type of N-linked glycosylation. The low amounts of genuine contaminating proteinsand other compounds provide for easy purification of secreted recombinant products. Since virus particles, pyrogens, pathogens, or cellderived components with an oncogenic potentialare not observed in these yeast species, reliaTable 3 Differences Among the Methylotrophic Yeasts H. polymorpha

P. pastoris

Mostly nonhomologous recombination Highcopy integration single-copy Mainly integration (up to 100 copies) MOX and FMD promoters as components for expressionplasmids FMD/MOX follow a repression/ derepression/induction mechanism mechanism One-step fermentationon glycerol or on glycerol/methanol possible glycerol Uraciland leucine-deficient

hosts

Homologous recombination AOX promoter as component for expressionplasmids AOX follows a repression/induction Two-step fermentation: growth on followed by induction with methanol Histidine-deficient host

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ble expression systemsare at hand that assure a high safetystandard for products considered for administration to humans. Ongoing risk assessment studies confirm the ecological safety of recombinants derived from H. polymorphs. In summary, the methylotrophs represent useful advanced alternatives for heterologous protein production and in many instances may provide the system of choice for the expression of a recombinant product.

ACKNOWLEDGMENTS We thank our co-workers J. Diirkop, H. Ervens, and P. Mackscheidt and our colleagues U. Weydemann, U. Dahlems, K. Melber, M. Piontek, P. Keup, F. Muller, and A. W.M. Strasser at Rhein Biotech GmbH, Diisseldorf for their valuable help and contributions to part of the experimental work reviewed in this chapter, and for their suggestions and support in finishing the manuscript.

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