Protein NMR Techniques (Methods in Molecular Biology, v831)

METHODS

IN

MOLECULAR BIOLOGY™

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

Protein NMR Techniques Third Edition Edited by

Alexander Shekhtman and David S. Burz Department of Chemistry, University at Albany, State University of New York, Albany, NY, USA

Editors Alexander Shekhtman Department of Chemistry University at Albany State University of New York 1400 Washington Avenue Albany, NY 12222, USA [email protected]

David S. Burz Department of Chemistry University at Albany State University of New York 1400 Washington Avenue Albany, NY 12222, USA [email protected]

ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-61779-479-7 e-ISBN 978-1-61779-480-3 DOI 10.1007/978-1-61779-480-3 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011943883 © Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)

Preface The field of protein NMR spectroscopy has rapidly expanded into new areas of biochemistry, molecular biology, and cell biology research that were impossible to study as recently as 10 years ago. The potential to study macromolecular systems that were once considered too large or too transient or too complex by using NMR spectroscopy is now being realized with the development of innovative technologies. Standard NMR technologies are also getting a facelift in part due to the pervasive nature of high-throughput approaches in biochemical and biomedical research. These advances warrant a new edition of Protein NMR Techniques that includes an authoritative but down-to-earth description of new methodologies. This edition consists of 24 chapters divided into four major categories: NMR sample preparation, solution NMR methodologies, solid-state NMR methodologies, and data processing. The material presented contains enough detail for use not only in specialized NMR laboratories, but in biochemical, molecular, and cell biology research labs that have access to high-field NMR spectrometers. Preparing proteins for NMR spectroscopy can be a time-consuming process that may take longer than data collection and analysis combined. Expression in bacterial cells still remains one of the most popular ways of preparing NMR samples. Chapter 1 discusses new methods for optimizing and increasing the production of isotope-labeled protein in bacteria. However, some proteins are difficult to express in bacteria, in these cases an alternative approach involves using yeast cells. Chapter 2 describes a methodology for producing proteins in yeast that are usually secreted into the growth medium. This technique is proving to be as robust and economic as bacterial production. One drawback to using proteins secreted by yeast is that they may exhibit altered patterns of glycosylation and phosphorylation. To avoid this problem and achieve proper posttranslational modifications, proteins are best produced in insect or mammalian cells. Advances in the use of these cells for producing NMR samples are detailed in Chapters 3 and 4. Cell-free expression of proteins has become a method of choice for high-throughput protein production especially in cases, where the yields from in vivo overexpression are very low. Cell-free systems allow for the selective incorporation of any isotope-labeled amino acid into a target protein with minimal scrambling. Chapters 5 and 6 describe the cell-free production of proteins for solution and solidstate NMR, respectively. Finally, although well-expressed in bacterial cells, some soluble proteins do not fold properly in sufficient quantity to permit analyses of structure, dynamics, and interactions. Chapter 7 presents a methodology for expressing and purifying such proteins in a cost-efficient manner. The chapters on solution NMR methodologies range from the study of individual proteins, large multidomain proteins, protein–ligand and protein–nucleic acid complexes in vitro, to the study of proteins inside living cells. A strategy for studying supramolecular systems, which has become possible due to advances in isotope labeling and NMR pulse sequences, is described in Chapter 8. Chapter 9 presents basic protocols and the latest improvements for measuring relaxation rates and analyzing protein dynamics. Methods to help overcome difficulties in applying solution NMR to the study of membrane proteins are

v

vi

Preface

detailed in Chapter 10. Structurally characterizing multi-domain proteins can be challenging due to the inherent flexibility present in these systems and requires special approaches outlined in Chapter 11. To regulate biological activity, proteins engage in interactions with other macromolecules present in the cell. A description of methods used to prepare protein–RNA, protein–DNA, and protein–ligand complexes suitable for study by using NMR spectroscopy are presented in Chapters 12, 13, and 14. Lastly, Chapter 15 describes in-cell NMR spectroscopy, a relatively new area of NMR research that affords atomic resolution information about isotope-labeled proteins inside living cells. Solid-state NMR spectroscopy presents a complementary approach to studying proteins, especially since the method is not limited by the molecular size constraints that hamper solution NMR. With the availability of high-field NMR spectrometers, solid-state NMR has become a viable technique for acquiring unique information about protein systems that are difficult to characterize by using solution NMR. Chapter 16 reviews the use of magic angle spinning solid-state NMR to study the structure and dynamics of perdeuterated proteins. The preparation and characterization of protein complexes for solid-state NMR and methodologies to analyze the structures and dynamics of protein complexes are presented in Chapter 17. The area of membrane protein expression has seen extensive advances of late, spurred by intense interest in signaling pathways, but impeded by difficulties in preparing samples in sufficient quantity for NMR spectroscopy. Chapter 18 details methods for producing membrane proteins suitable for study by using solid-state NMR. Processing and analyzing NMR data has historically been an extremely laborious part of NMR research, requiring skillful NMR spectroscopists to assign chemical shifts and to determine atomic resolution structures of proteins. With the advent of high-throughput assignment protocols, this task has become largely manageable by a trained graduate student. Nevertheless, there are difficult cases for which there is no substitution for the experienced spectroscopist. For example, characterization of eukaryotic kinases by NMR spectroscopy is complicated by the extensive dynamics and large size exhibited by these proteins. Chapter 19 describes the procedures used to assign backbone resonances for ERK2. The reactivity of solvent-exposed backbone amides varies by a factor of at least a billion-fold because of electrostatic interactions at the protein surface. The use of electrostatic analysis of hydrogen exchange rates to analyze protein flexibility is reviewed in Chapter 20. Chapter 21 presents a strategy for assigning the backbone resonances of small- to medium-sized globular proteins in a few hours by using a highly automated program, BATCH, to acquire, process, and analyze NMR data. A versatile protocol, UNIO, that provides nearly fully automated structure determination is described in Chapter 22. In UNIO, user-intervention is encouraged and facilitated by graphical tools for preparing, analyzing, validating, and presenting the NMR structure. Chapter 23 details the use of the ARIA software, which incorporates both solution and solid-state NMR structural constraints to perform structure calculations. The final chapter, Chapter 24, introduces the software DYNAMICS for analyzing relaxation rates that characterize the overall tumbling and local dynamics of a protein. This book presents a comprehensive description of the latest innovations in the field of protein NMR. It focuses on the importance of biochemistry, molecular biology, and cell biology to NMR spectroscopy while avoiding excessive repetition of existing material, which is readily available through a number of excellent texts and reviews that cover topics

Preface

vii

relevant to studying proteins by using NMR. Rather than reiterating the fundamental principles behind NMR methodologies, we have emphasized the practical aspects of experimental design combined with practical advice and examples. We hope that this book will provide both experienced NMR spectroscopists and biochemists, who are new to the field of NMR, with enough background to successfully apply these techniques to their research. Albany, NY, USA

Alexander Shekhtman David S. Burz

Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v xi

1 A Novel Bacterial Expression Method with Optimized Parameters for Very High Yield Production of Triple-Labeled Proteins . . . . . . . . . . . . . . . . . . . 1 Victoria Murray, Yuefei Huang, Jianglei Chen, Jianjun Wang, and Qianqian Li 2 Isotopic Labeling of Heterologous Proteins in the Yeast Pichia pastoris and Kluyveromyces lactis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Toshihiko Sugiki, Osamu Ichikawa, Mayumi Miyazawa-Onami, Ichio Shimada, and Hideo Takahashi 3 Isotope Labeling in Insect Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Krishna Saxena, Arpana Dutta, Judith Klein-Seetharaman, and Harald Schwalbe 4 Isotope Labeling in Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Arpana Dutta, Krishna Saxena, Harald Schwalbe, and Judith Klein-Seetharaman 5 Cell-Free Protein Production for NMR Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Mitsuhiro Takeda and Masatsune Kainosho 6 Cell-Free Membrane Protein Expression for Solid-State NMR . . . . . . . . . . . . . . . . 85 Alaa Abdine, Kyu-Ho Park, and Dror E. Warschawski 7 Expression and Purification of Src-family Kinases for Solution NMR Studies . . . . . . 111 Andrea Piserchio, David Cowburn, and Ranajeet Ghose 8 NMR Studies of Large Protein Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Shiou-Ru Tzeng, Ming-Tao Pai, and Charalampos G. Kalodimos 9 Protein Dynamics by 15N Nuclear Magnetic Relaxation . . . . . . . . . . . . . . . . . . . . . . 141 Fabien Ferrage 10 Bacterial Production and Solution NMR Studies of a Viral Membrane Ion Channel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 Jolyon K. Claridge and Jason R. Schnell 11 Preparation of the Modular Multi-Domain Protein RPA for Study by NMR Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 Chris A. Brosey, Marie-Eve Chagot, and Walter J. Chazin 12 NMR Studies of Protein–RNA Interactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Carla A. Theimer, Nakesha L. Smith, and May Khanna 13 Preparation and Optimization of Protein–DNA Complexes Suitable for Detailed NMR Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 My D. Sam and Robert T. Clubb

ix

x

Contents

14 NMR Studies of Protein–Ligand Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 Michael Goldflam, Teresa Tarragó, Margarida Gairí, and Ernest Giralt 15 In-Cell NMR Spectroscopy in Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 Kirsten E. Robinson, Patrick N. Reardon, and Leonard D. Spicer 16 Deuterated Peptides and Proteins: Structure and Dynamics Studies by MAS Solid-State NMR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 Bernd Reif 17 Solid-State NMR Spectroscopy of Protein Complexes . . . . . . . . . . . . . . . . . . . . . . . 303 Shangjin Sun, Yun Han, Sivakumar Paramasivam, Si Yan, Amanda E. Siglin, John C. Williams, In-Ja L. Byeon, Jinwoo Ahn, Angela M. Gronenborn, and Tatyana Polenova 18 Synthesis, Purification, and Characterization of Single Helix Membrane Peptides and Proteins for NMR Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 Miki Itaya, Ian C. Brett, and Steven O. Smith 19 Assignment of Backbone Resonances in a Eukaryotic Protein Kinase – ERK2 as a Representative Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359 Andrea Piserchio, Kevin N. Dalby, and Ranajeet Ghose 20 Electrostatics of Hydrogen Exchange for Analyzing Protein Flexibility . . . . . . . . . . 369 Griselda Hernández, Janet S. Anderson, and David M. LeMaster 21 Fast Protein Backbone NMR Resonance Assignment Using the BATCH Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407 Bernhard Brutscher and Ewen Lescop 22 Comprehensive Automation for NMR Structure Determination of Proteins . . . . . . 429 Paul Guerry and Torsten Herrmann 23 ARIA for Solution and Solid-State NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453 Benjamin Bardiaux, Thérèse Malliavin, and Michael Nilges 24 Determining Protein Dynamics from 15N Relaxation Data by Using DYNAMICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485 David Fushman Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

513

Contributors ALAA ABDINE • CNRS and Université Paris Diderot, IBPC, Paris, France JINWOO AHN • Department of Structural Biology, Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA JANET S. ANDERSON • Department of Chemistry, Union College, NY, USA BENJAMIN BARDIAUX • NMR-supported Structural Biology, Leibnitz-Institut für Molekulare Pharmakologie (FMP), Berlin, Germany IAN C. BRETT • Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA CHRIS A. BROSEY • Departments of Biochemistry and Chemistry, Center for Structural Biology, Vanderbilt University, Nashville, TN, USA BERNHARD BRUTSCHER • Institut de Biologie Structurale – Jean-Pierre Ebel, CNRS, CEA, UJF, UMR5075, Grenoble Cedex, France IN-JA L. BYEON • Department of Structural Biology, Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA MARIE-EVE CHAGOT • Departments of Biochemistry and Chemistry, Center for Structural Biology, Vanderbilt University, Nashville, TN, USA WALTER J. CHAZIN • Departments of Biochemistry and Chemistry, Center for Structural Biology, Vanderbilt University, Nashville, TN, USA JIANGLEI CHEN • Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI, USA JOLYON K. CLARIDGE • Department of Biochemistry, University of Oxford, Oxford, UK ROBERT T. CLUBB • Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA DAVID COWBURN • Departments of Biochemistry and Physiology & Biophysics, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY, USA KEVIN N. DALBY • Division of Medicinal Chemistry, University of Texas, Austin, TX, USA; Graduate Programs, Cellular and Molecular Biology, Pharmacy, Biomedical Engineering and Biochemistry, University of Texas, Austin, TX, USA ARPANA DUTTA • Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA FABIEN FERRAGE • Département de chimie, Ecole normale supérieure et Laboratoire des Biomolécules, CNRS UMR 7203, Paris, Cedex, France DAVID FUSHMAN • Department of Chemistry and Biochemistry and Center for Biomolecular Structure and Organization, University of Maryland, MD, USA MARGARIDA GAIRÍ • Servicios Cientifico Tecnicos, Universitat de Barcelona, Barcelona, Spain RANAJEET GHOSE • Department of Chemistry, The City College of New York, New York, NY, USA; Graduate Center of the City University of New York, New York, NY, USA

xi

xii

Contributors

ERNEST GIRALT • Institute for Research in Biomedicine (IRB Barcelona), Parc Científic de Barcelona, Barcelona, Spain; Departament de Química Orgànica, Universitat de Barcelona, Barcelona, Spain MICHAEL GOLDFLAM • Institute for Research in Biomedicine (IRB Barcelona), Parc Científic de Barcelona, Barcelona, Spain ANGELA M. GRONENBORN • Department of Structural Biology, Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA PAUL GUERRY • Centre Européen de RMN à très Hauts Champs, Université de Lyon, Ecole Normale Supérieure de Lyon, CNRS, Université Claude, Villeurbanne, France YUN HAN • Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, USA GRISELDA HERNÁNDEZ • Department of Health and Department of Biomedical Sciences, Wadsworth Center, School of Public Health, University at Albany – SUNY, Albany, NY, USA TORSTEN HERRMANN • Centre Européen de RMN à très Hauts Champs, Université de Lyon, CNRS, Ecole Normale Supérieure de Lyon, Université Claude, Villeurbanne, France YUEFEI HUANG • Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI, USA OSAMU ICHIKAWA • Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan MIKI ITAYA • Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA MASATSUNE KAINOSHO • Graduate School of Science, Nagoya University, Nagoya, Japan; Center for Priority Areas, Tokyo Metropolitan University, Hachioji, Japan CHARALAMPOS G. KALODIMOS • Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, USA MAY KHANNA • Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA JUDITH KLEIN-SEETHARAMAN • Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA DAVID M. LEMASTER • Department of Health and Department of Biomedical Sciences, Wadsworth Center, School of Public Health, University at Albany – SUNY, Albany, NY, USA EWEN LESCOP • Laboratoire de Chimie et Biologie Structurales, Institut de Chimie des Substances Naturelles, Centre de Recherche de Gif, CNRS, Gif-sur-Yvette, France QIANQIAN LI • Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI, USA THÉRÈSE MALLIAVIN • Unité de Bioinformatique Structurale, CNRS URA 2185, Institut Pasteur, Paris, France MAYUMI MIYAZAWA-ONAMI • Japan Biological Informatics Consortium (JBiC), Tokyo, Japan; Biomedicinal Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan

Contributors

xiii

VICTORIA MURRAY • Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI, USA MICHAEL NILGES • Unité de Bioinformatique Structurale, CNRS URA 2185, Institut Pasteur, Paris, France MING-TAO PAI • Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, USA SIVAKUMAR PARAMASIVAM • Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, USA KYU-HO PARK • CNRS and Université Paris Diderot, IBPC, Paris, France ANDREA PISERCHIO • Department of Chemistry, The City College of New York, New York, NY, USA TATYANA POLENOVA • Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, USA PATRICK N. REARDON • Department of Biochemistry, Duke University NMR Center, Durham, NC, USA BERND REIF • Munich Center for Integrated Protein Science (CIPSM) at Department Chemie, Technische Universität München, Garching, Germany; Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin, Germany; Helmholtz-Zentrum München (HMGU), German Research Center for Environmental Health, Neuherberg, Germany KIRSTEN E. ROBINSON • Department of Biochemistry, Duke University NMR Center, Durham, NC, USA MY D. SAM • Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA KRISHNA SAXENA • Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University Frankfurt, Frankfurt am Main, Germany JASON R. SCHNELL • Department of Biochemistry, University of Oxford, Oxford, UK HARALD SCHWALBE • Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University Frankfurt, Frankfurt am Main, Germany ICHIO SHIMADA • Biomedicinal Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan; Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan AMANDA E. SIGLIN • Department of Molecular Medicine, Beckman Research Institute of City of Hope, Duarte, CA, USA NAKESHA L. SMITH • Department of Chemistry, University at Albany SUNY, Albany, NY, USA STEVEN O. SMITH • Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA LEONARD D. SPICER • Department of Biochemistry, Duke University NMR Center, Durham, NC, USA; Department of Radiology, Duke University NMR Center, Durham, NC, USA TOSHIHIKO SUGIKI • Japan Biological Informatics Consortium (JBiC), Tokyo, Japan; Biomedicinal Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan

xiv

Contributors

SHANGJIN SUN • Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, USA HIDEO TAKAHASHI • Department of Supramolecular Biology, Graduate School of Nanobioscience, Yokohama City University, Yokohama, Japan; Biomedicinal Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan MITSUHIRO TAKEDA • Graduate School of Science, Nagoya University, Nagoya, Japan TERESA TARRAGÓ • Institute for Research in Biomedicine (IRB Barcelona), Parc Científic de Barcelona, Barcelona, Spain CARLA A. THEIMER • Department of Chemistry, University at Albany SUNY, Albany, NY, USA SHIOU-RU TZENG • Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, USA JIANJUN WANG • Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI, USA DROR E. WARSCHAWSKI • CNRS and Université Paris Diderot, IBPC, Paris, France JOHN C. WILLIAMS • Department of Molecular Medicine, Beckman Research Institute of City of Hope, Duarte, CA, USA SI YAN • Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, USA

Chapter 1 A Novel Bacterial Expression Method with Optimized Parameters for Very High Yield Production of Triple-Labeled Proteins Victoria Murray, Yuefei Huang, Jianglei Chen, Jianjun Wang, and Qianqian Li Abstract The Gram-negative bacterium Escherichia coli offer a means for rapid, high-yield, and economical production of recombinant proteins. However, when preparing protein samples for NMR, high-level production of functional isotopically labeled proteins can be quite challenging. This is especially true for the preparation of triple-labeled protein samples in D2O (2H/13C/15N). The large expense and time-consuming nature of triple-labeled protein production for NMR led us to revisit the current bacterial protein expression protocols. Our goal was to develop an efficient bacterial expression method for very high-level production of triple-labeled proteins that could be routinely utilized in every NMR lab without changing expression vectors or requiring fermentation. We developed a novel high cell-density IPTG-induction bacterial expression method that combines tightly controlled traditional IPTG-induction expression with the high cell-density of auto-induction expression. In addition, we optimize several key experimental protocols and parameters to ensure that our new high cell-density bacterial expression method routinely produces 14–25 mg of triple-labeled proteins and 15–35 mg of unlabeled proteins from 50-mL bacterial cell cultures. Key words: High yield protein production, Bacterial expression, Isotopic labeling, NMR

1. Introduction To perform NMR structural studies of proteins, we have to produce proteins that are isotopically labeled with 13C and 15N for small proteins (<20 kDa) and with 13C, 15N, and 2H for larger proteins (>20 kDa). Among the many systems available for heterologous protein production, the Gram-negative bacterium Escherichia coli remains one of the most attractive hosts (1, 2). This is especially true for isotopically labeling proteins since bacterial expression Alexander Shekhtman and David S. Burz (eds.), Protein NMR Techniques, Methods in Molecular Biology, vol. 831, DOI 10.1007/978-1-61779-480-3_1, © Springer Science+Business Media, LLC 2012

1

2

V. Murray et al.

provides the cheapest way to prepare these proteins for NMR studies (3). Protein expression and purification is a routine practice in many NMR labs, but it is not uncommon to see a drastic reduction in protein yield when isotopically labeling the proteins, especially when D2O must be used. To overcome these difficulties, we have developed a novel bacterial expression method that combines the tightly controlled traditional IPTG-induction bacterial expression with the high celldensity of the auto-induction method (4). To summarize our procedure, we first determine how to make a proper starting culture, followed by double colony selection and finally high cell-density expression. With these optimized protocols and parameters, our new bacterial expression method offers near gram quantity production of triple-labeled proteins from one-liter bacterial cell cultures, without changing expression vectors and without fermentation (5). Thus, every NMR laboratory can easily apply this novel bacterial expression method on a routine basis for the production of a very high yield of triple-labeled proteins for their NMR structural studies of proteins.

2. Materials 2.1. Sample Preparation and SDS-PAGE

1. 4× SDS loading buffer: 200 mM Tris–HCl, pH 6.8, 8% (w/v) sodium dodecyl sulfate (SDS), 0.4% (w/v) bromophenol blue, 40% glycerol. Store at room temperature. 2. Dithiothreitol (DTT): Prepare 1 M solution using sterile water. Filter through a 0.22-μm pore size membrane (syringe filter) and store 1-mL aliquots at −20°C. 3. 30% Acrylamide/bis solution (29:1). 4. 1.5 M Tris–HCl, pH 8.8. 5. 10% SDS. 6. 10% ammonium persulfate. 7. TEMED. 8. M Tris–HCl, pH 6.8. 9. Protein molecular weight markers. 10. 5× SDS running buffer: 0.5% SDS, 125 mM Tris base, 1.25 M glycine. Store at room temperature. Do not adjust pH. 11. Coomassie blue-staining solution: 0.25% (w/v) Coomassie brilliant blue, 45% methanol, 10% acetic acid. Add brilliant blue to methanol and stir for 60 min. Add water and acetic acid and stir for another 30 min. Store at room temperature (see Note 1). 12. Destaining solution: 30% methanol, 10% acetic acid. Store at room temperature.

1

2.2. Protein Expression

A Novel Bacterial Expression Method with Optimized Parameters…

3

1. LB medium (Miller): Dissolve 25 g of powdered LB medium in 1 L of distilled water or D2O. Adjust the pH to 7.4 using NaOH. Autoclave and store at room temperature (see Note 2). Add antibiotics (KAN or AMP) prior to use. 2. Kanamycin monosulfate stock solution (KAN): Dissolve KAN monosulfate to a concentration of 30 mg/mL in distilled water. Syringe filter and store 1 mL aliquots at −20°C. 3. Ampicillin sodium sulfate stock solution (AMP): Dissolve AMP sodium sulfate to a concentration of 50 mg/mL in distilled water. Syringe filter and store 1 mL aliquots at −20°C. 4. LB agar plates (Miller): Dissolve 40 g of LB agar (Miller) in 1 L of distilled water and/or D2O in a 2-L flask. Cover with foil and autoclave. Monitor the temperature as it cools. When the temperature reaches ~50°C, add 1 mL of the KAN or AMP stock solution. Pour ~10 mL into 100 × 10-mm Petri dishes and swirl to coat the plate. Let the LB agar solidify at room temperature. Place plates back into a plastic bag, seal with tape, and store at 4°C (see Note 2). 5. Isopropyl-β-D-thiogalactopyranoside (IPTG): Prepare a 1 M solution using distilled water. Syringe filter and store 1 mL aliquots at −20°C. 6. 100% Glycerol: Autoclave to sterilize. Store at room temperature. 7. 5× M9 Salts (1 L): Dissolve 64 g of Na2HPO4, 15 g of KH2PO4, 5 g of NH4Cl, and 2.5 g of NaCl in distilled water or D2O, adjust the volume to 1 L. Autoclave to sterilize and store at room temperature (see Note 2). Omit NH4Cl from this recipe when 5× M9 salts are used for isotope-labeling. Do not adjust pH. 8. 20% Glucose: Dissolve 20 g glucose in distilled water, adjust volume to 100 mL. Sterilize by filtration and store at 4°C. 9. MgSO4: Prepare a 1-M solution, autoclave, and store at room temperature. 10. CaCl2: Prepare 1-M solution, autoclave, and store at room temperature. 11. M9 minimal medium for traditional IPTG method and double colony selection (100 mL): 78 mL of distilled, sterilized water, 20 mL of 5× M9 salts, 2 mL of 20% glucose, 200 μL of 1 M MgSO4, 10 μL of 1 M CaCl2, and 100 μL of antibiotic. Add the CaCl2 last and immediately swirl the flask to dissolve the cloudy precipitate. Adjust the pH to 7.4 using NaOH. 12. M9 minimal medium for high cell-density IPTG-induction method (100 mL): 75 mL of distilled, sterilized water, 20 mL of 5× M9 salts, 5 mL of 20% glucose, 200 μL of 1 M MgSO4, 10 μL of 1 M CaCl2, and 100 μL of antibiotic. Add the CaCl2 last and immediately swirl the flask to dissolve the cloudy precipitate (see Note 3). Adjust the pH to 7.4 using NaOH (see Note 4).

4

V. Murray et al.

13. M9 for double-labeling (100 mL): 80 mL of distilled, sterilized water, 20 mL of 5× M9 salts without NH4Cl, 100 mg of 15 NH4Cl and 0.2 g of 13C-glucose (for traditional IPTG method) or 1 g of 13C-glucose (for high cell-density IPTGinduction method), 200 μL of 1 M MgSO4, 10 μL of 1 M CaCl2, and 100 μL of antibiotic. Add the CaCl2 last and immediately swirl the flask to dissolve the cloudy precipitate (see Note 3). Adjust the pH to 7.4 using NaOH (see Note 4). Use a filtration unit with a 0.22-μm pore size to sterilize the medium (see Note 5). 14. M9 for triple-labeling (100 mL): 80 mL of 99% D2O, 20 mL of 5× M9 salts without NH4Cl in D2O, 100 mg of 15NH4Cl, and 0.2 g of 13C/2H-glucose (for traditional IPTG-induction method), or 1 g of 13C/2H-glucose (for high cell-density IPTG-induction method), 200 μL of 1 M MgSO4, 10 μL of 1 M CaCl2, and 100 μL of antibiotic. You can also use 13 C-glucose; however, this usually generates ~90% deuterated triple-labeled protein samples. Add the CaCl2 last and immediately swirl the flask to dissolve the cloudy precipitate (see Note 3). Adjust the pH to 7.4 using NaOH (see Note 4). Use a filtration unit with a 0.22-μm pore size to sterilize the medium (see Note 5). 15. 1000× Trace metals (100 mL): Dissolve 811 mg of FeCl3 (50 mM), 222 mg of CaCl2 (20 mM), 125.8 mg of MnCl2 (10 mM), 161.5 mg of ZnSO4 (10 mM), 26 mg of CoCl2 (2 mM), 26.9 mg of CuCl2 (2 mM), 25.9 mg of NiCl2 (2 mM), 41.2 mg of Na2MoO4 (2 mM), 34.6 mg of Na2SeO3 (2 mM), and 12.4 mg of H3BO3 (2 mM) in 60 mM HCl. Autoclave to sterilize. Store at room temperature. 16. BME vitamins (see Note 3). 2.3. Protein Purification

1. Affinity resin: His-Bind Resin. 2. 8× Charge buffer: 400 mM NiSO4. Store at 4°C. 3. 8× Binding buffer: 160 mM Tris–HCl, pH 7.9, 2 M NaCl, 20 mM imidazole. Store in an amber bottle at room temperature. 4. 8× Wash buffer: 160 mM Tris–HCl, pH 7.9, 2 M NaCl, 240 mM imidazole. Store in an amber bottle at room temperature. 5. 4× Elute buffer: 80 mM Tris–HCl, pH 7.9, 1 M NaCl, 4 M imidazole. Store in an amber bottle at room temperature. 6. (NH4)2CO3: Dissolve 474.4 g of (NH4)2CO3 in 3.5 L of water. Once dissolved, adjust the volume to 4 L (final concentration 1.23 M) and store at room temperature. Do not adjust pH. 7. Urea.

1

A Novel Bacterial Expression Method with Optimized Parameters…

5

3. Methods When triple-labeling proteins, bacteria have to be grown in D2O, usually causing a significant reduction in protein yields. We sought to overcome this obstacle. Our strategy mainly focuses on increasing the cell density of bacterial expression without manipulation of the expression vector or use of a fermenter. Unfortunately, bacterial expression at a high cell-density in D2O usually causes several major problems, including (1) plasmid loss, (2) significant reduction in the pH of the growth medium due to cell metabolites, and (3) limited availability of dissolved oxygen. These problems often result in a low or even no protein production with high cell-density bacterial expression. We developed several practical protocols that solved these problems, including (1) preparation of a proper starting culture, (2) double colony selection in D2O, (3) optimization of bacterial expression conditions, and (4) better control of the pH of the medium. We further developed a high cell-density IPTG-induction bacterial expression method that combines the tightly controlled traditional IPTG-induction expression with high cell-density autoinduction expression. Our optimized protocols ensure plasmid stability inside bacterial cells, resulting in routine production of 14–25 mg triple-labeled proteins from a 50-mL bacterial cell culture. Importantly, this novel bacterial expression method uses the same expression vectors as the traditional IPTG-induction method and does not require a fermenter. Thus, every NMR laboratory can easily adopt this novel bacterial expression method to produce large quantities of triple-labeled proteins. 3.1. PAGE Sample Preparation

PAGE samples come either from the cell lysate isolated immediately after bacterial expression or from the column flow through during protein purification. 1. For samples collected directly from bacterial cell culture: Collect 500 μL of cells and place in a 1.7-mL microcentrifuge tube. Spin down at 12,000 × g for 5 min at room temperature using a microcentrifuge, discard the supernatant, and tap out the excess on a paper towel. 2. Add 25 μL of 4× SDS loading buffer and 25 μL of water and resuspend the pellet. Store the samples in a freezer until ready to run a gel. 3. Before running the culture samples on a gel, place the samples on a 90°C heat block for 30 min. Remove from the heat block; add 50 μL of water and vortex for 30 s (see Note 6). 4. For samples collected from flow through during protein purification, mix 60 μL of column flow through with 20 μL of

6

V. Murray et al.

4× SDS loading buffer and mix thoroughly by repeated pipetting up and down. Store the samples in a freezer until ready to run a gel. 5. Before running the flow through samples on a gel, place the samples on a 90°C heat block for 5 min (see Note 6). 6. Before loading samples on a gel, centrifuge at 12,000 × g for 10 min at room temperature using a microcentrifuge to pellet cellular debris (see Note 7). 3.2. SDS-PAGE

1. Depending on the protein size, choose an appropriate acrylamide percentage for the resolving gel (see Note 8). 2. To prepare a 10% SDS-PAGE mini-resolving gel (5 mL), using a mini-gel apparatus: Mix 1.9 mL of water, 1.7 mL of 30% acrylamide/bis solution, 1.3 mL of 1.5 M Tris–HCl, pH 8.8, 50 μL of 10% SDS, 50 μL of 10% ammonium persulfate, and 2 μL of TEMED. Mix well and pour between glass plates set in a loading cassette. Leave about 1.5-cm space on top for the stacking gel. Gently pipet water on top and let the gel set (about 20 min). 3. Pour a stacking gel (2 mL) once the resolving gel has set. Mix 1.4 mL of water, 330 μL of 30% acrylamide/bis solution, 250 μL of 1 M Tris–HCl, pH 6.8, 20 μL of 10% SDS, 20 μL of ammonium persulfate, and 2 μL of TEMED. Pour the water off the top of the resolving gel, remove excess water with filter paper, and pour the stacking gel. Insert a comb containing the appropriate number of lanes and let the stacking gel set (about 20 min). 4. Prepare 1× running buffer by diluting 100 mL of 5× running buffer solution with 400 mL of distilled water. Make sure to mix the solution well. Pour the buffer into the inner and outer chambers of the gel apparatus. For cell culture samples, load 7.5 μL of each sample into the lanes. For column flow through samples, load 20 μL into each lane. Make sure to load 5 μL of molecular weight markers in one lane. 5. Secure the lid on the gel box and plug into a power supply. Run the gel at 88 V for ~2 h. Turn off the power supply when the blue dye front reaches the bottom of the gel. 6. Remove the gel from the glass plates and place it in a small box with 20 mL of Coomassie blue-staining solution. Allow the gel to stain for at least 30 min (see Note 1). Pour out the stain, rinse the gel with water to remove excess stain and then add 30 mL of destaining solution. Place a small piece of paper towel in the box to accelerate the destaining process. This process may take a few hours; however, you can start to detect bands within 30–60 min.

1

3.3. A Proper Starting Culture

A Novel Bacterial Expression Method with Optimized Parameters…

7

A critical consideration for high-level bacterial expression is the preparation of a proper starting culture in a rich medium for scaling up in minimal medium. The general practice in NMR laboratories is to grow an overnight culture using a rich medium, such as LB, at 37°C. We observe that an overnight culture usually reaches saturation by the next morning, and may result in plasmid instability and loss due to several factors including basal leakage of the T7 expression system that expresses the toxic target proteins to the host cells under this overgrowth condition (4, 6, 7). This usually results in a poor yield of target protein. Figure 1 shows a growth curve for E. coli BL21 (DE3) cells carrying the LCAT/pET30a vector, in H2O-based LB, suggesting that the bacteria are in the exponential or log phase of growth between 6 and 7.5 h at 37°C (see Note 9). We placed particular emphasis on double colony selection (Subheading 3.5) to ensure that a high percentage of bacterial cells within this colony contain the DNA expression plasmid. 1. Perform a time course of bacterial growth of a new protein expression vector in rich (LB) medium by measuring the OD600 every 30 min for ~10–12 h in water-based rich medium and ~16–18 h in D2O-based rich medium (see Note 10). The OD600

Fig. 1. Plot of E. coli growth in 5 mL of LB medium over a 10-h period at 37°C starting with a glycerol stock. The bacterial strain, BL-21(DE3), contains a pET30a vector expressing the gene for lecithin:cholesterol acyltransferase (LCAT). Based on this plot, the log phase of the culture is between an OD600 of 1 and 3.5. Note: The growth curve is vector, protein, and bacterial strain dependent. Reproduced from Murray 2010 with permission from Cold Spring Harbor.

8

V. Murray et al.

of the log phase and the time to reach saturation are vector, protein, and bacterial strain-dependent; therefore, we suggest performing this experiment before actually expressing protein for purification and labeling (see Note 11). 2. Once the optimal OD600 of the log phase of the starting culture is determined, this will be the OD600 for all future starting cultures using both traditional IPTG expression and high celldensity expression methods. 3.4. Traditional IPTG Method

This method can be scaled up or down to suit your needs. This following protocol is used to make a 100-mL expression culture. 1. Prepare a 5-mL proper starting culture as described in Subheading 3.3 in a 50-mL conical tube with holes poked in the lid. 2. Prepare 100 mL of M9 minimal medium in a 250-mL flask. Add 1.5 mL of the starting culture and measure the OD600. (To check the OD600 at this point, aliquot 1 mL of the cell culture into a cuvette and measure the OD600.) We suggest a starting OD600 between 0.05 and 0.10 for healthy bacterial cell growth. Place the flask in a 37°C incubator with a shaking speed of 200 rpm. 3. Start to monitor the OD600 after 3 h (to check the OD600 at this point, dilute 100 μL of cell culture into a cuvette containing 900 μL of distilled water and measure the OD600. The final OD600 value is ten times the spectrophotometer reading). Once the OD600 reaches between 0.8 and 1.2 (see Note 12), remove 2 mL of cell culture and place it in a 15-mL culture tube (for a noninduced reference). Induce the remaining culture with 0.5 mM IPTG. Place both the flask and the 15-mL culture tube in a 20°C incubator overnight with a shaking speed of 200 rpm. 4. The following morning, measure the final OD600 of the cell culture. If the protein expression is induced at an OD600 of 1, the OD600 of the bacterial cell culture will be around 2–3, indicating healthy bacterial cell growth. Harvest the cells by spinning down at 10,000 × g for 10 min at 4°C using a benchtop centrifuge. Remove the supernatant and either store the cell pellet at −80°C or use immediately for protein purification. 5. To check protein expression levels, take 500 μL samples of each culture (non-induced and IPTG-induced), follow the sample preparation protocol (Subheading 3.1) and run an SDS-PAGE (Subheading 3.2) to compare non-induced and IPTG-induced samples.

3.5. Double Colony Selection

Since plasmid loss is encountered during bacterial expression in D2O much more frequently than in H2O, we describe a double colony selection procedure for triple-labeling protein in D2O. Based on our

1

A Novel Bacterial Expression Method with Optimized Parameters…

9

Fig. 2. SDS-PAGE of protein expression of apoE(1–215)/pTYB1 in D2O before (a), during (b) and after (c) double colony selection. Arrows indicate the expected protein band (~80 kDa). (a) Shows four different colonies before colony selection. (b) Shows the results of three different colonies selected from the single colony selection (lanes 1–3) and another three colonies selected from the double colony selection (lanes 4–6). The second colony selection was based on Colony 3 (lane 3, b). (c) Shows the results of six colonies from the double colony selection, indicating a high level of protein expression for all six colonies. Reproduced from Sivashanmugam 2009 with permission from Wiley Interscience.

experience, this is a critical protocol that significantly increases the yield of triple-labeled proteins. Figure 2 shows the result of a typical double colony selection of apoE(1–215)/pTYB1 in D2O, demonstrating that high protein expression levels are achieved after double colony selection. This high-level expression of apoE(1–215)/pTYB1 in D2O has been stable for more than 2 years. Usually, we perform double colony selection before we optimize the expression conditions. Thus, the traditional IPTGinduction method is the default method with which to start double colony selection. The procedure, described in Subheading 3.4, can be applied here for double colony selection, except for culture volume and the use of D2O. 1. Prepare LB agar plates with 50% D2O. 2. Perform a bacterial transformation using LB agar plates prepared with D2O. 3. Next afternoon, choose nine colonies to make starting cultures of 5 mL of LB in 50% D2O and 5 μL of antibiotic in 50-mL conical tubes, and prepare a master plate (see Note 13). Punch holes in the lids of the tubes. Eight of the nine colonies will be induced with IPTG and the ninth colony will be used as a negative, non-induced control. 4. When the starting culture is ready, add 50–100 μL to 5 mL of M9 minimal medium in 70% D2O and 5 μL of antibiotic in 50-mL conical tubes. Ensure that the starting OD600 is between 0.05 and 0.10. Place the tube in a 37°C incubator with a shaking speed of 200 rpm.

10

V. Murray et al.

5. When the OD600 of the culture reaches between 0.8 and 1 (see Note 14), add 0.5 mM IPTG to eight of the nine cultures to induce protein expression, clearly marking which culture is serving as a negative control. Place the tubes in a 20°C incubator overnight with a shaking speed of 200 rpm. 6. The following morning, remove 500 μL of cell culture from each tube and place in a microcentrifuge tube. Centrifuge at 12,000 × g for 10 min at room temperature and discard the supernatant. Prepare SDS-PAGE samples and run a gel. 7. Choose the colony expressing the biggest protein band (using the negative control as reference) and prepare 5 mL of LB culture in either 70 or 99% D2O and 5 μL of antibiotic containing the colony from the master plate. Grow at 37°C until the OD600 reaches 0.7–0.9 and spread 150 μL on an LB plate prepared in 50% D2O. Invert the plate and incubate it at 37°C overnight. 8. The following day, repeat steps 3–6 using 70 or 99% D2O for the second round of colony selection (see Note 14). When completed, choose the colony expressing the biggest protein band to make a 5-mL LB culture in 100% D2O and 5 μL of antibiotic. Grow the culture halfway through its log phase. Add 800 μL of culture to 200 μL of 100% sterile glycerol in a 2-mL cryogenic tube with a screw top cap. Pipet up and down to mix thoroughly and flash freeze the tubes by dipping them in liquid nitrogen, store at −80°C. If the protein expression level is extremely high, we suggest that you make at least 5–10 glycerol stocks of this double selected colony for future use. 3.6. High Cell-Density IPTG-Induction Method

This is a hybrid method combining traditional IPTG-induction and auto-induction bacterial expression methods. It takes advantage of tightly controlled IPTG-induction and the high cell-density of the auto-induction bacterial expression. We use rich media, such as LB and 2× YT, to reach a high cell-density before IPTGinduction and then switch the culture medium by gently spinningdown the cells and resuspending them into an equal volume of minimal medium. However, many problems may occur during a high cell-density bacterial expression that can cause a significant reduction in protein yield, such as reduced pH of the expression medium, poor aeration, and/or plasmid loss during expression. We describe the following procedure to avoid these problems, ensuring a high yield of triple-labeled protein. 1. Using the glycerol stock prepared after double colony selection (Subheading 3.5), make a starting culture in 50 mL of LB medium containing 50 μL antibiotic in a 250-mL flask (Dip the pipet tip in the glycerol stock and scratch the surface. Place the tip into the culture medium, pipet up and down a few

1

A Novel Bacterial Expression Method with Optimized Parameters…

11

times and remove the tip. Immediately return the glycerol stock to the −80°C freezer). Place the flask in a 37°C incubator with a shaking speed of 200 rpm and incubate until the OD600 is halfway through the log phase (Subheading 3.3, step 1). DO NOT let the starting culture grow overnight since the saturation of cell growth may cause plasmid loss. 2. Transfer the cells into sterile tubes and spin down the culture at 5,000 × g for 7 min at room temperature. Remove the supernatant and tap the tubes on paper towels to remove as much LB as possible. 3. Gently resuspend the pellets in 50 mL of M9 minimal medium (see Note 4) and transfer the resuspended cells to a 250-mL sterile flask. Place the flask in an incubator that is set at the optimal induction temperature (see Subheading 3.7). Maintain the shaking speed at 200 rpm for efficient aeration and keep the culture at the optimal temperature for 1–1.5 h to allow the cells to adapt to the new medium. An enhancement of approximate 0.5–1 U of OD600 should be observed at the end of this time. For example, if the OD600 is 3 right after medium exchange, you can expect the OD600 to reach 3.5–4 at the end of the 1–1.5 h incubation, indicating healthy cell growth (this increase in OD600 will be slightly less when expressing in D2O). 4. Add the optimal concentration of IPTG (Subheading 3.7) to induce protein expression and keep the culture in the incubator for an optimal period of time (Subheading 3.7) at a shaking speed of 200 rpm. 5. Measure the final OD600 before harvesting cells. You can expect to see a two- to fourfold enhancement at the end of the expression. For example, if your OD600 is 4 after the 1–1.5 h incubation, the final OD600 will be between 8 and 16, indicating healthy bacterial growth. 6. Harvest the cells by spinning down the culture at 10,000 × g for 10 min at 4°C. Remove the supernatant and either store the cell pellet at −80°C, or immediately use for protein purification. 3.7. Optimization of Various Conditions

Another important step for high-level protein production using high cell-density bacterial expression is to optimize the expression conditions such as culture temperature and the induction time. These steps are critical for the initial expression of a protein using the high cell-density expression method. We usually use the traditional IPTG-induction method first to check if a new protein can be expressed by using bacteria. Once the protein expression is confirmed by the traditional IPTG-induction method, we can optimize the high cell-density expression method to produce high-yield isotopically labeled proteins.

12

V. Murray et al.

We usually carry out time courses at different temperatures, such as 15, 20, room temperature, 30 and 37°C. We normally prepare a 5-mL starting culture either in D2O or in water for the time course and closely monitor the following parameters during expression: OD600, pH, and target protein production. The detailed procedure of optimization follows: 1. Prepare 10 mL starting cultures in 50-mL flasks: 10 mL of LB medium in 99% D2O, 10 μL of antibiotic and bacterial cells from a glycerol stock after double colony selection. Incubate at 37°C with a shaking speed of 200 rpm. 2. Once the optimal OD600 has been reached, gently centrifuge the cells at 5,000 × g for 7 min at room temperature and discard the supernatant. Resuspend the cell pellets with 10 mL of M9 minimal medium in 99% D2O in 50-mL flasks and incubate at various temperatures, such as 15, 20, room-temperature, 30° and 37°C, for 1 h. Check the OD600 of the cultures before and after this 1-h cell incubation. 3. If the OD600 of each culture after the 1-h incubation increases by 0.5–1 OD600 units, this indicates that the cells are healthy and growing after the medium exchange. Induce protein expression by adding 0.5 mM IPTG. Be sure to choose one culture without IPTG-induction to serve as the negative control. Return the tubes to appropriate shakers at different temperatures. We found that 0.5 mM IPTG usually gives a reasonable protein production, thus we always used this IPTG concentration as our starting point. However, an independent optimization of IPTG concentration can be carried out and is discussed in step 6 of this section. 4. For cultures growing below 25°C, let them grow overnight (~14–16 h). The following morning, collect 500 μL of cell culture samples every 2 h (typically collect between 16 and 28 h and one more sample the next morning). For the cultures growing above 25°C, start to collect samples every 2 h after induction for at least 8 h and one the next morning. At each time point, check the OD600 and the pH of the cell culture (see Note 4). Be sure to keep the collected cell pellets at −20°C. 5. When all of the samples have been collected, prepare samples for SDS-PAGE analysis. A comparison with the negative control (noninduced culture) allows you to determine which temperature and induction time give you the best protein yield (see Note 15). 6. Repeat the above procedure to optimize the IPTG concentration using the optimized temperature and incubation time. We usually test IPTG concentrations of 0.1, 0.25, 0.5, 0.75, and 1 mM. 7. Once the optimal conditions have been determined, you can now perform high cell-density bacterial expression on a larger

1

A Novel Bacterial Expression Method with Optimized Parameters…

13

scale. Be sure to use a large flask for better aeration. We usually use a 250-mL flask for a 50-mL cell culture and a 500-mL flask for a 100-mL cell culture. If you want to grow a 200-mL cell culture, we suggest dividing the culture into 2 × 100 mL cultures in two 500-mL flasks. Figure 3 shows a typical time course experiment performed during optimization of experimental conditions. Table 1 shows the expression parameters for the time course during our optimization of the expression of human apolipoprotein A-I (apoAI). It is clear that at the maximum OD600 the bacterial expression produces the highest yield of triple-labeled apoAI, as confirmed by a Western blot (Fig. 3).

Fig. 3. Left panel: An SDS-PAGE showing auto-induction time course of triple-labeled human apoAI expression in D2O at room temperature. Lanes 1–7 = 24, 28, 32, 36, 40, 44, and 54 h, respectively. Right panel is a Western blot of the same time course using anti-apoAI monoclonal antibody. Reproduced from Sivashanmugam 2009 with permission from Wiley Interscience.

Table 1 Parameters of the time course of human apolipoprotein A–I expression Time

24 h

28 h

32 h

36 h

40 h

44 h

54 h

OD600

2.5

3.9

7.2

9.1

8.4

8

8.1

pH

6.6

6.5

6.3

6

6

6

6.1

Protein yield

−

+

++

+++

++

++

−

14

V. Murray et al.

3.8. Protein Purification

Protein purification depends on the fusion tag that is used, as different tags serve different purposes. In our laboratory, we generally use histidine tags. In this section, we describe a typical protein purification procedure using a His-Bind Resin column. 1. Prepare 1× dilutions of all buffers. Recheck the pH to ensure that they are still 7.9. 2. Centrifuge the cell culture at 10,000 × g for 10 min at 4°C. Remove the supernatant and resuspend the pellets in 20 mL of 1× binding buffer. If the protein is in inclusion bodies and can be refolded readily during dialysis, you can resuspend the pellet in 20 mL of 1× binding buffer containing 6 M urea. 3. Lyse the cells by using either sonication or a French press. Centrifuge the lysate at 16,000 × g for 20 min at 4°C. Collect the supernatant and store it on ice. 4. Add 10 mL of 1× binding buffer and repeat step 2 at least twice, and then combine all the supernatants. Depending on the protein, you may need to add additional binding buffer and repeat step 2 3–5 times to completely extract the protein from the cells. 5. Equilibrate the affinity column with 50 mL of 1× charge buffer (see Note 16). Remove the charge buffer and equilibrate the column with 50 mL of 1× binding buffer. The column should be a light blue color after equilibration. 6. Load the column with the clear lysate from step 3. The flow rate should be ~1 mL/minute (see Note 17). Collect the flow through and remove a 60-μL sample for SDS-PAGE. 7. Wash the column with 200 mL of 1× binding buffer, followed by an additional 100 mL of 1× wash buffer. The flow rate of wash buffer should also be ~1 mL/min. Elute the column with 100 mL of 1× elution buffer. Collect the last drop of elution for SDS-PAGE to make sure that all of the protein has been eluted from the column. 8. Perform SDS-PAGE analysis with all of the collected samples to assess the purification. 9. Place the eluted protein into a dialysis bag and dialyze extensively against water containing 20 mM (NH4)2CO3 to remove imidazole, salts and possibly urea. After dialysis, freeze the protein sample with liquid nitrogen and lyophilize to obtain pure triple-labeled protein powder. Run a gel to assess the purity of the protein powder.

3.9. Conclusion

With the high cell-density IPTG-induction bacterial expression method and the practical protocols described above, we routinely produce 14–25 mg of triple-labeled proteins and 15–35 mg of unlabeled proteins from a 50-mL cell culture for all the proteins we tested. Table 2 lists the final yields of unlabeled and triplelabeled proteins obtained using high cell-density bacterial expression

1

A Novel Bacterial Expression Method with Optimized Parameters…

15

Table 2 Final yields of unlabeled and triple labeled proteins: high cell-density vs. traditional IPTG method Protein

High cell densityb (mg)

IPTGb (mg)

M.W. (Cal) (Da)

M.W. (MS) (Da)

%Dc

Triple-labeled RAP(1–210) RAP(91–323) ApoE(1–183)a Mouse apoAI(1–216) Human apoAI

20 ± 3 25 ± 3 18 ± 4 15 ± 2 14 ± 1

0.5 0.8 2 0.8 0.6

33,801 36,633 22,866 28,014 32,814

33,525 ± 195 36,376 ± 200 22,686 ± 116 27,732 ± 125 32,401 ± 150

~92 ~93 ~89 ~90 ~88

Unlabeled Human apoAI Human apoE

34 ± 1 17 ± 2

1 0.2

M.W. molecular weight Reproduced from Sivashanmugam 2009 with permission from Wiley Interscience a ApoE(1–183) was expressed in 40% D2O, the rest are expressed in 99.7% D2O b High cell density (50-mL culture volume): high cell-density expression methods, including auto-induction and high cell-density IPTG-induction; IPTG: the optimized traditional IPTG-induced expression. We repeated the expressions at least three times for all proteins, the yield shown is the average ± standard deviation c Estimated percentage of deuteration, assuming 100% 13C and 15N-labeling. For apoE(1–183), the %D is the estimated percentage of deuteration based on 40% D2O. For the other four proteins, the %D is the estimated percentage of deuteration based on 99.7% D2O

compared with the yields obtained by using the traditional IPTGinduction method in a 50-mL cell culture, the results suggest a 5–100-fold enhancement in protein yield. In addition, the protocols described produce a consistent high-level of triple-labeled protein, which is always reproducible. Table 2 also gives mass spectroscopic data for the triple-labeled protein, indicating the efficiency of deuteration for triple-labeled protein using auto-induction expressions. Overall, the deuteration efficiency is around 90% if we assume the 13C and 15N-labeling are 100%. This is because we used 99.7% D2O and nondeuterated 13C-glycerol or 13C-glucose in high celldensity expressions. This result is comparable to the deuteration efficiency of the traditional IPTG-induction expression method using single labeled 13C-glucose.

4. Notes 1. The stain can be reused multiple times. When using fresh stain, you only need to stain gels for 15–30 min. Pour the used stain into a separate container. When reusing stain, you may need to stain gels longer.

16

V. Murray et al.

2. When using D2O to replace water in LB agar, broth or M9 salts, solutions CANNOT be autoclaved to sterilize. When making LB broth, follow the directions as stated, but instead of autoclaving, use a filtration unit with a 0.22-μm pore size to sterilize. When making LB agar plates, use a microwave to bring the solution just to a boil (to dissolve agar), add antibiotics once the agar temperature cools to ~50°C, and pour into plates. When making 5× M9 salts, follow the directions as stated, however omit NH4Cl for isotopic-labeling. Once again, do not autoclave; sterilize using a filtration unit with a 0.22-μm pore size. 3. For the auto-induction method, Studier suggests using vitamins and trace metals (4). We found vitamins and trace metals help promote healthy bacterial growth when using our high cell-density IPTG-induction bacterial expression. We purchased the trace metals and BME vitamins stock solution from Sigma. The trace metals used in our laboratory is based on Studier’s recipe provided in the supplement materials of his elegant paper on the auto-induction bacterial expression method (4). For our optimized high cell-density IPTGinduction minimal medium, we added 0.25× vitamins and 0.25× trace metals (see Table 1 in ref. 5). 4. While monitoring OD600 during the optimization time course (Subheading 3.7), the pH should be monitored as well. As the cell density increases, the pH of the culture lowers due to the release of cell metabolites. If the pH becomes too low (pH < 6), it will affect bacterial cell health and protein production. If the pH drops below 6 during the time course, we increase the pH of the M9 minimal medium to 8 using NaOH to allow for a larger buffering capacity of the culture medium. 5. M9 minimal medium containing isotopes (2H, 13C, or 15N) cannot be autoclaved. It must be sterilized using a filtration unit containing a 0.22-μm pore size. 6. If the protein contains cysteine residue(s), add 10–20 mM DTT after adding water. Vortex the sample well and let it sit for 30–60 min at room temperature. 7. The release of DNA can cause the sample to become quite viscous, making it hard to load on the gel. If you notice this, you can simply sonicate your sample at a low wattage (4–6 W) for 5–10 s. Afterward, spin down the sample at 12,000 × g for 2 min at room temperature. Also, when loading the gel, remove the sample from the top portion of the supernatant to avoid the pelleted cellular debris at the bottom. 8. To determine which percentage gel to use, follow the guidelines found in Table A8-8 in Sambrook and Russell (8). Also, if you are working with proteins that weigh less than 15 kDa,

1

A Novel Bacterial Expression Method with Optimized Parameters…

17

12% Tricine gels or gradient gels are highly recommended for better resolution in the molecular weight range of 5–15 kDa. 9. Bacteria display a four-phase pattern of cell growth in liquid media. First, there is an initial lag phase when bacteria are adapting to the growth conditions; at this point, an increase in OD600 will not be seen. Second, bacteria enter their exponential or log phase at which point the bacterial cells start dividing (doubling in number). The OD600 during this log phase climbs steadily. Third, bacteria enter the stationary phase during which the rate of cell growth significantly slows due to a decrease in available nutrients and an accumulation of toxins. The OD600 will level off during this phase. Finally, if fresh medium is not made available and toxins are not removed, bacteria will enter the death phase and a noticeable drop in OD600 will be observed. The key is to utilize a starting culture during the exponential or log phase of their growth curve (see Fig. 1). 10. When performing a time course using rich media, such as LB or 2× YT in D2O, bacteria grow much more slowly than in water. 11. Based on our experience, we suggest the appropriate OD600 range for the starting culture is between 3 and 5 in LB medium and between 5 and 7 in 2× YT medium. However, the OD600 of the log phase is vector, protein, and bacterial strain-dependent. Thus, the best way to determine the middle point of the log phase is to perform a time course of bacterial growth for each new protein expression vector. 12. Based on our experience, for healthy bacterial cell growth, the culture is expected to reach an OD600 of 0.8–1.2 within 4–6 h for bacterial expressions in water and within 6–9 h for bacterial expressions in D2O if the starting OD600 is between 0.05 and 0.10. 13. When performing colony selection, it is helpful to make a “master plate.” Take a KAN or AMP plate and make a 9-square grid under the agar plate. Label boxes 1–9. When you are ready to inoculate the medium, take a sterilized tip, gently touch the selected colony, and then gently touch the agar of the master plate in the corresponding box. Go back to the original plate, retouch the same colony, and drop it into a tube. Repeat this procedure for all selected colonies. Once finished, put the lid back on the plate, invert and incubate at 37°C for about 8 h. Colonies should be about 1–2-mm in diameter. Cut a long strip of parafilm, wrap the edges of the plate, and store inverted at 4°C. You can use this plate to regrow the colonies for future cell cultures. However, the plate is only good for about 2 weeks, so be sure to also make glycerol stocks. 14. If the bacteria do not grow well in 70% D2O, then they must be trained to adapt to D2O medium. For this purpose, pick a colony off a D2O plate and start a 5-mL bacterial culture of LB

18

V. Murray et al.

medium in 25% D2O. Once the OD600 of the culture reaches 1 at 37°C, transfer 100 μL of the cell culture into 5 mL of LB medium in 50% D2O. The starting OD600 of this new culture is about 0.1. Let the cell culture grow at 37°C until the OD600 reaches 1 and transfer 100 μL of the cell culture into 5 mL of LB medium in 75% D2O and let the culture to grow at 37°C until the OD600 reaches 2–3. Use this cell culture as your starting culture. 15. A Western blot of this time course will further allow an unambiguous determination of the time point that produces the best protein yield. 16. Typically, we prepare columns containing 5 mL of affinity resin for a 50-mL cell culture since 2.5 mL of resin can bind about 20 mg of protein. This can be scaled up or down to suit your needs based on the expected protein yield. 17. If the flow rate is too slow, we have found that using a 1.5-μm syringe filter to remove cellular debris from the clear lysate prevents columns from becoming clogged and running slow. References 1. Swartz, J.R. (2001) Advances in Escherichia coli production of therapeutic proteins. Curr. Opin. Biotechnol. 12, 195–201. 2. Hewitt, L., and McDonnell, J.M. (2004) Screening and optimizing protein production in E. coli methods. Methods Mol. Biol. 278, 1–16. 3. McIntosh, L.P. and Dahlquist, F.W. (1990) Biosynthetic incorporation of 15N and 13C for assignment and interpretation of nuclear magnetic resonance spectra of proteins. Q Rev. Biophys. 23, 1–38. 4. Studier, F.W. (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234. 5. Sivashanmugam, A., Murray, V., Cui, C., Zhang, Y., Wang, J., and Li, Q. (2009) Practical

protocols for production of very high yields of recombinant proteins using Escherichia coli. Protein Sci. 18, 936–948. 6. Chen, H.C., Hwang, C.F., and Mou, D.G. (1992) High-density Escherichia coli cultivation process for hyperexpression of recombinant porcine growth hormone. Enzyme Microb. Technol. 14, 321–326. 7. Baneyx, F. (1999) Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 10, 411–421. 8. Sambrook, J. and Russell, D. (2001) Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (ISBN 978087969577-4).

Chapter 2 Isotopic Labeling of Heterologous Proteins in the Yeast Pichia pastoris and Kluyveromyces lactis Toshihiko Sugiki, Osamu Ichikawa, Mayumi Miyazawa-Onami, Ichio Shimada, and Hideo Takahashi Abstract Several protein expression systems are available for the preparation of stable isotope-labeled recombinant proteins for NMR studies. Yeast expression systems have several advantages over prokaryotic systems, such as the widely used Escherichia coli expression system. Protein expression using the methylotrophic yeast Pichia pastoris is commonly employed for the preparation of isotope-labeled proteins. Recently, the hemiascomycete yeast Kluyveromyces lactis expression system was reported as being useful for preparing proteins for NMR studies. Since each yeast expression system has different features, their applications have increased in number. In this chapter, we describe procedures for the efficient production of uniformly isotope-labeled proteins using the P. pastoris and the K. lactis yeast expression systems. Key words: Yeast expression systems, Pichia pastoris, Kluyveromyces lactis, Stable isotope labeling, Fed-batch fermentation, NMR

1. Introduction The most widely used method for the expression of isotopically labeled heterologous recombinant proteins is the Escherichia coli expression system because of easy handling, rapid and high-density cell growth, high levels of protein production, and relatively low costs for isotope-labeling. However, in many cases, expression of structurally and functionally intact eukaryotic proteins by E. coli and other prokaryotic cells is fundamentally difficult due to (1) the lack of intracellular organelles, (2) a limited number of molecular chaperones, and (3) the absence of posttranslational modification mechanisms (1–6). Yeast, however, combines several advantages of

Alexander Shekhtman and David S. Burz (eds.), Protein NMR Techniques, Methods in Molecular Biology, vol. 831, DOI 10.1007/978-1-61779-480-3_2, © Springer Science+Business Media, LLC 2012

19

20

T. Sugiki et al.

both eukaryotic and prokaryotic expression systems (1) processing, folding, complex disulfide-bond network formation, and posttranslational modification (e.g., glycosylation) of proteins are possible, (2) yeast exhibit rapid growth rates and grow to high-density, and (3) methods for molecular genetic manipulation are well established and are simple to perform (1–6). Here, we introduce the Pichia pastoris and Kluyveromyces lactis yeast expression systems for the stable isotopic labeling of heterologous proteins. 1.1. The P. pastoris Expression System

Host cells and expression vectors are available from Invitrogen. Based upon Invitrogen instruction manuals (7), previous reports (8–15), and our experience, we describe here optimized cell culture procedures for the production of isotope-labeled heterologous proteins by P. pastoris. P. pastoris is capable of utilizing methanol as both a carbon source and to induce protein expression. Methanol is oxidized by alcohol oxidases, AOX1 and AOX2, in yeast cell peroxisomes (though the majority of alcohol oxidase activity is attributed to AOX1). The AOX1 promoter tightly regulates expression of the AOX1 gene, and methanol induces AOX1 promoter activity. Thus, the AOX1 promoter is used to drive the expression of a target protein by replacing the AOX1 gene with a cDNA encoding the desired heterologous protein (1–6). Invitrogen supplies several vector series that are commonly used for protein expression in P. pastoris, including pPIC9K, pPIC3.5K, and pPICZ. Using these vectors, a cDNA cassette containing the target gene under the control of the AOX1 promoter is inserted into the genome of P. pastoris by using homologous recombination along with a gene coding for resistance to a drug, such as Zeocin™ or G418, for subsequent selection of transformed cells (7). Target proteins are expressed either intracellularly or secreted into the medium by P. pastoris. For secretion of target proteins, vectors pPICZα and pPIC9K are recommended (7). The pPICZα and pPIC9K series contain a gene that encodes a prepro signal sequence [such as the Saccharomyces cerevisiae α-mating factor (α-MF)] between the AOX1 promoter and the target gene (7). Although target proteins may contain native secretion signals, in many cases, the α-MF sequence is generally used as the sole secretion signal. Since P. pastoris secretes a low amount of its native proteins, a secreted target protein comprises the vast majority of the total proteins in the culture medium, simplifying purification of the target protein (1–7). A different set of vectors lacking secretion signal sequences are required if the target protein is to be expressed intracellularly. For cytosolic and nonglycosylated proteins, vectors pPICZ or pPIC3.5K are recommended (7). In this chapter, we describe methods and provide technical advice for producing uniformly labeled [U-13C, 15N] and [U-2H, 15N] target proteins in P. pastoris (8–14). The procedures described are

2

Isotopic Labeling of Heterologous Proteins in the Yeast…

21

optimized for secretory expression of isotope-labeled DDR2 from P. pastoris strain X-33 (Mut+ phenotype transformants) using the pPICZα vector system (15). 1.2. The K. lactis Expression System

Host cells and expression vectors are available from New England Biolabs. Based upon instruction manuals (16), previous reports, and our experience, we describe here optimized cell culture procedures for the production of isotope-labeled heterologous proteins by K. lactis. Heterologous recombinant proteins expressed by K. lactis can be sequestered intracellularly or secreted into the medium. As with P. pastoris, K. lactis secretes very low levels of native proteins, therefore secreted target proteins comprise the vast majority of the total protein present in the culture medium, which simplifies purification of the target protein (16–18). In K. lactis, the LAC4 promoter drives expression of the target protein gene. Using the pKLAC vector, a cDNA cassette containing the target gene under the control of the LAC4 promoter is inserted into the genome of K. lactis by using homologous recombination (16–18). The transcriptional activity of the LAC4 promoter is induced by galactose, which K. lactis also utilizes as a carbon source for cell growth. Therefore, expression of the target protein is constitutively induced by cultivation in a medium containing galactose (16–18). The pKLAC vectors impart a fungal acetamidase gene (amdS) to the transformants. Since only transformants that express amdS can utilize acetamide as a sole nitrogen source, amdS is used for selection of the desired transformants, thereby obviating the need for expensive antibiotics, such as Zeocin™ and rendering this auxotrophic selection method more cost-effective (16–18). In addition, auxotrophic acetamide selection enriches populations of transformants in which multiple tandem copies of target cDNA are integrated into the yeast genome (16–18). Thus, the K. lactis protein expression system is simple, cost-effective, easily scaled-up, and highly reproducible. We recently established a cost-effective isotope-labeling method utilizing the hemiascomycete yeast K. lactis (6, 19). In the most commonly employed K. lactis expression system, 20 g/L galactose is required as a carbon source for cell growth and as an inducer of target protein expression (16). However, while a 20 g/L carbon source is acceptable for uniform 15N-labeling, it is economically infeasible for uniform 13C-labeling. Sugiki et al. reported that by combining K. lactis strain GG799, which is characterized by weak glucose suppression for the LAC4 promoter (16), with a fed-batch culture method, larger amounts of protein can be expressed using a smaller amount of glucose, thus reducing costs to a level comparable to isotope-labeling by E. coli systems (6, 19). In this chapter, we describe methods and provide technical advice for producing uniformly labeled [U-13C, 15N] and [U-2H, 15N] target proteins in K. lactis.

22

T. Sugiki et al.

2. Materials The recipes for the culture media described here were derived mainly from the manuals for the EasySelect™ Pichia Expression Kit (Invitrogen), the Pichia Fermentation Process Guidelines (Invitrogen), and the K. lactis Protein Expression Kit (New England Biolabs). Autoclave sterilization is performed at 121°C for 15 min. 2.1. Uniform 13C, 15 N-Labeling Using P. pastoris

1. 40% unlabeled D-glucose stock solution: Dissolve 200 g of D-glucose in 1 L of water. Sterilize by aseptic filtration and store at room temperature. The shelf life of this solution is approximately 1 year. 2. 100 mg/mL Stock solution of Zeocin™ (Invitrogen). 3. Yeast extract peptone dextrose sorbitol (YPDS) agar plates: Dissolve 20 g of bacto peptone, 10 g of yeast extract, 182.2 g of sorbitol, and 20 g of bacto agar in 950 mL of water. Sterilize by autoclaving and cool to 50–60°C. Aseptically add 1 mL of 100 mg/mL Zeocin™ and 50 mL of 40% glucose stock solution, gently mix well, and dispense into sterile disporsable Petri dishes. Store at 4°C in the dark. The shelf life of these agar plates is 1–2 weeks. 4. 10× Yeast nitrogen base (YNB): Dissolve 34 g of YNB without amino acids and ammonium sulfate in 1 L of water (see Note 1). Warm it to 40–50°C to dissolve completely if needed. Sterilize by aseptic filtration and store at 4°C. The shelf life of this solution is approximately 1 year. 5. 1 M Potassium phosphate: Mix 132 mL of 1 M K2HPO4 and 868 mL of 1 M KH2PO4. Adjust the pH to 6.0 ± 0.1 using KOH or phosphoric acid. Sterilize by autoclaving and store at room temperature. The shelf life of this solution is greater than 1 year. 6. 500× Biotin: Dissolve 20 mg of biotin in 100 mL of water and warm it to 40–50°C to dissolve completely. Sterilize by aseptic filtration and store at 4°C. The shelf life of this solution is approximately 1 year. 7. 10% (w/v) Glycerol: Dissolve 50 mL of glycerol in 450 mL of water. Sterilize by aseptic filtration and store at room temperature. The shelf life of this solution is over 1 year. 8. 200× PTM1 trace salts: Mix together the following ingredients and dissolve to a final volume of 1 L in water: 6.0 g of CuSO 4⋅5H2O, 0.08 g of NaI, 3.0 g of MgSO4⋅H2O, 0.2 g of Na2MoO4⋅2H2O, 0.02 g of boric acid, 0.5 g of CoCl2, 20.0 g of ZnCl2, 65.0 g of FeSO4⋅7H2O, 0.2 g of biotin, 5.0 mL of H2SO4. Warm to 40–50°C to dissolve completely if needed. Sterilize by aseptic filtration and store at 4°C or room temperature. The shelf life of this solution is approximately 3 months at 4°C.

2

Isotopic Labeling of Heterologous Proteins in the Yeast…

23

9. Buffered glycerol-complex (BMGY) medium: Dissolve 20 g bacto peptone and 10 g yeast extract in 700 mL of water. Sterilize by autoclaving and cool it to room temperature. Aseptically add 100 mL of sterile 1 M potassium phosphate, 100 mL of sterile 10× YNB, 2 mL of sterile 500× biotin, and 100 mL of sterile 10% (w/v) glycerol solutions. If needed, 5 mL of 200× PTM1 trace salts solution can be added to this medium. Store at 4°C. The shelf life of this medium is approximately 3 months. 10. 10% 15N-ammonium chloride: Dissolve 6 g of 15N-ammonium chloride (98 atom% 15N) in 60 mL of water (see Note 2). Sterilize by aseptic filtration. Prepare this solution just before use. 11.

15

N-BM medium: To prepare 1.2 L of uniformly 15N-labeled buffered minimal (15N-BM) medium, autoclave 900 mL of water in a 2-L media bottle and allow the water to cool to room temperature. Aseptically add 120 mL of 10× YNB, 2.4 mL of 500× biotin, 120 mL of 1 M potassium phosphate, and 60 mL of 10% 15N-ammonium chloride to the autoclaved water. If needed, 6 mL of 200× PTM1 trace salts solution can be added to 15N-BM medium.

12. 5% D-[13C6]glucose: Dissolve 1 g of D-[13C6]glucose (99 atom% 13 C) in 20 mL of 15N-BM medium and sterilize by aseptic filtration. Prepare this solution just before use. 13. Antifoaming agent: (see Note 3). 14.

13

C-methanol (99 atom% 13C).

15. 50% (w/v) ethanol. Ethanol is just necessary for immersing air outlet port of the fermentation system. 2.2. Uniform 2H, 15 N-Labeling Using P. pastoris

1. YPDS agar plates (Subheading 2.1). 2. BMGY medium (Subheading 2.1). 3. 10× YNB(2H2O): Prepare this solution in a manner similar to 10× YNB solution (Subheading 2.1), but use 2H2O (99.8 atom % 2H) instead of water (H2O). Sterilize by aseptic filtration and store at room temperature. The shelf life of this solution is approximately 2–3 months. 4. 1 M potassium phosphate(2H2O): Prepare 150 mL of this solution in a manner similar to 1 M potassium phosphate solution (Subheading 2.1), but use 2H2O instead of water. Sterilize by aseptic filtration. Prepare this solution just before use. 5. 500× biotin(2H2O): Prepare this solution in a manner similar to 500× biotin solution (Subheading 2.1), but use 2H2O instead of water. Sterilize by aseptic filtration and store at 4°C. The shelf life of this solution is approximately 2–3 months. 6. 200× PTM1 trace salts(2H2O): Prepare this solution in a manner similar to 200× PTM1 trace salts solution (Subheading 2.1), but use 2H2O instead of water. Sterilize by aseptic filtration and store at 4°C.

24

T. Sugiki et al.

7. 10% 15N-ammonium chloride(2H2O): Dissolve 7 g of 15 N-ammonium chloride in 70 mL of 2H2O (see Note 2) and sterilize by aseptic filtration. Prepare this solution just before use. 8. 10% unlabeled D-glucose(2H2O): Dissolve 1 g of D-glucose in 10 mL of 2H2O and sterilize by aseptic filtration. Prepare this solution just before use. 9. 15N-BMD (90% 2H2O) medium: To prepare 10 mL of uniformly 15 N-labeled buffered minimal medium containing unlabeled glucose prepared in 90% 2H2O, mix 6 mL of 2H2O and 1 mL of H2O, and sterilize by aseptic filtration. Aseptically add 1 mL of 10× YNB(2H2O), 0.02 mL of 500× biotin(2H2O), 1 mL of 1 M potassium phosphate(2H2O), 0.5 mL of 10% unlabeled D-glucose(2H2O) (0.5% final concentration), 0.5 mL of 10% 15 N-ammonium chloride(2H2O), and 5–10 μL of antifoaming agents. If needed, 0.05 mL of 200× PTM1 trace salts(2H2O) can be added to this medium. Prepare this medium just before use. 10.

15

N-BM(2H2O) medium: To prepare 1.3 L of uniformly N-labeled buffered minimal medium in 100% 2H2O, sterilize 956 mL of fresh 2H2O by aseptic filtration. Aseptically add 130 mL of 10× YNB(2H2O), 3 mL of 500× biotin(2H2O), 130 mL of 1 M potassium phosphate(2H2O), and 66 mL of 10% 15N-ammonium chloride(2H2O) into the sterile 2H2O. If needed, 7 mL of 200× PTM1 trace salts(2H2O) can be added to this medium. Prepare this medium just before use. 15

11.

15

N-BMD(2H2O) medium: To prepare 5 mL of uniformly N-labeled buffered minimal medium containing unlabeled glucose in 100% 2H2O, aseptically add 0.25 mL of 10% unlabeled D-glucose(2H2O) to 4.75 mL of 15N-BM(2H2O) in a 50-mL Corning tube. Prepare this medium just before use. 15

12. 10% D-[2H7]glucose(2H2O): Dissolve 1.5 g of D-[2H7]glucose (98 atom% 2H) in 15 mL of 2H2O and sterilize by aseptic filtration. Prepare this solution just before use. 13. 2H, 15N-BMD(2H2O) medium: To prepare 300 mL of uniformly 15 N-labeled buffered minimal medium containing D-[2H7]glucose in 100% 2H2O, transfer 285 mL of 15N-BM(2H2O) into a sterilize, well-dried 1-L baffled flask. Aseptically add 15 mL of 10% D-[2H7]glucose(2H2O) and 0.15–0.30 mL of antifoaming agents. Prepare this medium just before use. 14. Antifoaming agent (Subheading 2.1). 15. [2H4]methanol (99.5 atom% 2H). 16. 50% (w/v) ethanol. Ethanol is just necessary for immersing air outlet port of the fermentation system. 2.3. Uniform 13C, 15 N-Labeling Using K. lactis

1. Yeast peptone (YP): Dissolve 20 g of bacto peptone and 10 g of yeast extract in 950 mL of water. Sterilize by autoclaving and store at room temperature or 4°C. The shelf life of this solution is approximately 1 year.

2

Isotopic Labeling of Heterologous Proteins in the Yeast…

25

2. 40% unlabeled D-glucose stock solution (Subheading 2.1). 3. YPD medium: Aseptically add 50 mL of sterile 40% D-glucose stock solution to the autoclaved 950 mL YP solution. The shelf life of this solution is approximately 1 month. 4. 1 M potassium phosphate (Subheading 2.1). 5. YCB-acetamide agar plates: Dissolve 5.85 g of yeast carbon base (YCB) medium powder and 10 g of bacto agar in 470 mL of water. Add 25 mL of 1 M potassium phosphate solution. Autoclave and cool it to 50–60°C. Aseptically add 5 mL of 100× acetamide stock solution (supplied by New England Biolabs), gently mix well, and dispense into sterile disporsable Petri dishes. Store at 4°C. The shelf life of these plates is approximately 3 months. 6. 10× YNB (Subheading 2.1). 7. 500× biotin (Subheading 2.1). 8. 200× PTM1 trace salts (Subheading 2.1). 9. 10% 15N-ammonium chloride: Dissolve 7.5 g of 15N-ammonium chloride in 75 mL of water (see Note 2) and sterilize by aseptic filtration. Prepare this solution just before use. 10. 4% D-[13C6]glucose: Dissolve 2 g of D-[13C6]glucose in 50 mL of water and sterilize it by aseptic filtration. Prepare this solution just before use. 11.

13

C, 15N-BMD medium: To prepare 0.5 L of 15N-BM medium containing 0.4% D-[13C6]glucose, autoclave 325 mL of water containing 0.25–0.50 mL of antifoaming agents in a 2-L fermentation vessel and allow it to cool to room temperature. Aseptically add 50 mL of 10× YNB, 1 mL of 500× biotin, 50 mL of 1 M potassium phosphate, 25 mL of 10% 15N-ammonium chloride, and 50 mL of 4% D-[13C6]glucose to the autoclaved water for a final volume of 500 mL. If needed, 2.5 mL of 200× PTM1 trace salts solution can be added to this medium.

12. 6% D-[13C6]glucose: Dissolve 6 g of D-[13C6]glucose in 100 mL of water and sterilize by aseptic filtration. Prepare this solution just before use. 13. 13C, 15N-BMD feeding medium: To prepare 1 L of 15N-BM medium containing 0.6% D-[13C6]glucose feeding medium, autoclave 650 mL of water containing 0.5–1.0 mL of antifoaming agents in a 1-L media bottle and allow it to cool to room temperature. Aseptically add 100 mL of 10× YNB, 2 mL of 500× biotin, 100 mL of 1 M potassium phosphate, 50 mL of 10% 15 N-ammonium chloride and 100 mL of 6% D-[13C6]glucose to the autoclaved water for a total volume of 1 L. If needed, 5 mL of 200× PTM1 trace salts solution can be added to this medium. 14. Antifoaming agent (Subheading 2.1). 15. 50% (w/v) ethanol. Ethanol is just necessary for immersing air outlet port of the fermentation system.

26

T. Sugiki et al.

2.4. Uniform 2H, 15 N-Labeling Using K. lactis

1. YPD medium (Subheading 2.3). 2. YCB-acetamide agar plates (Subheading 2.3): If needed, prepare the plates with 2H2O (see Note 4). 3. 10× YNB(2H2O) (Subheading 2.2). 4. 500× biotin(2H2O) (Subheading 2.2). 5. 1 M potassium phosphate(2H2O) (Subheading 2.2). 6. 200× PTM1 trace salts(2H2O) (Subheading 2.2). 7. 10% 15N-ammonium chloride(2H2O): Dissolve 7.5 g of 15 N-ammonium chloride in 75 mL of 2H2O (see Note 2) and sterilize by aseptic filtration. Prepare this solution just before use. 8. 4% D-[2H7]glucose(2H2O): Dissolve 2 g of D-[2H7]glucose in 50 mL of 2H2O and sterilize by aseptic filtration. Prepare this solution just before use. 9. 2H, 15N-BMD(2H2O): To prepare 0.5 L of uniformly 15 N-labeled buffered minimal medium containing D-[2H7]glucose in 2H2O, sterilize 325 mL of 2H2O by aseptic filtration. Aseptically add 50 mL of 10× YNB(2H2O), 1 mL of 500× biotin(2H2O), 50 mL of 1 M potassium phosphate(2H2O), 25 mL of 10% 15N-ammonium chloride(2H2O), 50 mL of 4% D-[2H7]glucose(2H2O), and 0.25–0.50 mL of antifoaming agent to the sterilized 2H2O for a final volume of 500 mL. If needed, 2.5 mL of 200× PTM1 trace salts(2H2O) can be added to this medium. 10. 6% D-[2H7]glucose(2H2O): Dissolve 6 g of D-[2H7]glucose in 100 mL of 2H2O and sterilize by aseptic filtration. Prepare this solution just before use. 11. 2H, 15N-BMD(2H2O) feeding medium: Sterilize 650 mL of 2 H2O by aseptic filtration. Aseptically add 100 mL of 10× YNB(2H2O), 2 mL of 500× biotin(2H2O), 100 mL of 1 M potassium phosphate(2H2O), 50 mL of 10% 15N-ammonium chloride(2H2O), 100 mL of 6% D-[2H7]glucose(2H2O), and 0.5–1.0 mL of antifoaming agent to the sterilized 2H2O for a final volume of 1 L. If needed, 5 mL of 200× PTM1 trace salts(2H2O) can be added to this medium. 12. Antifoaming agent (Subheading 2.1). 13. 50% (w/v) ethanol. 13. 50% (w/v) ethanol. Ethanol is just necessary for immersing air outlet port of the fermentation system.

3. Methods 3.1. Uniform 13C, 15 N-Labeling Using P. pastoris

1. Prepare necessary media. (a) Using aluminum foil, loosely cover the open lid of an empty 1-L baffled flask and autoclave it.

2

Isotopic Labeling of Heterologous Proteins in the Yeast…

27

(b) Prepare 1.2 L of fresh, sterile 15N-BM medium in a 2-L medium bottle and 20 mL of 5% D-[13C6]glucose. (c) Aseptically combine 180 mL of fresh 15N-BM medium, 20 mL of 5% D-[13C6]glucose and 0.1–0.2 mL of antifoaming agents in the sterile 1-L baffled flask to make 13 C, 15N-BMD medium. (d) Using aluminum foil, loosely mask the open lid of the empty 2-L fermentation vessel. Clamp all of the lines that could come into contact with the growth medium and autoclave the vessel. (e) Aseptically combine 1 L of fresh 15N-BM medium, 0.5–1.0 mL of antifoaming agent and 5 mL of 13C-methanol into the sterile empty 2-L fermentation vessel, to make 13 C, 15N-BMM medium. 2. Using a sterile loop, scoop a small aliquot of P. pastoris transformants from a frozen glycerol stock, and streak it onto a YPDS agar plate containing Zeocin™. Incubate the plate for 24–48 h at 30°C. 3. To produce the primary culture, inoculate a fresh, single colony of the P. pastoris transformants into 5 mL of BMGY medium (in a 50-mL Corning tube) and shake at 200–250 rpm at 30°C for 18–24 h. 4. Pellet the primary culture cells by centrifugation at 3,000 × g for 5 min at 20°C and discard the supernatant. Gently resuspend the pellet with 30 mL of fresh, sterile 13C, 15N-BMD medium, and pour the resuspended cells into the remaining 13 C, 15N-BMD medium in the baffled flask for a final volume of 200 mL (see Note 5). Shake the flask at > 200 rpm at 30°C until the cell density reaches an OD600 of 4–6. 5. Pellet the cells in sterile tubes by centrifuging at 2,000 × g for 10 min at 20°C and discard the supernatant. Gently resuspend the pellet with 30 mL of fresh, sterile 13C, 15N-BMM medium, and pour the resuspended cells into the remaining fresh, sterile 13 C, 15N-BMM medium in the 2-L fermentation vessel to reach an OD600 of approximately 1 (see Notes 6–8). 6. Assemble the fermentation system. Aseptically attach the pH and dissolved O2 sensors to the fermentation vessel and connect the probes to their respective controllers. Connect the outlet port of the air-feeding tube to the air flow inlet port of the vessel. Aseptically attach a Liebig condenser to the outlet port of the vessel, and immerse the air-exhaust port of the Liebig condenser under 1 L of 50 % (w/v) ethanol solution. Insert the temperature probe into the vessel and connect it to a temperature controller, such as a circulating water bath. 7. Agitate the culture medium at 300–800 rpm with 0.1–0.3 L/ min of feeding air at 30°C (see Notes 9 and 10). Add

28

T. Sugiki et al. 13

C-methanol (0.5 % (w/v) of the total volume) to the culture medium every 24 h during this induction phase (20) (see Note 11). The total amount of 13C-methanol required for sufficient protein expression in a cost-effective manner is approximately 10–20 mL per 1 L culture medium (see Note 12). 8. Pellet the cells by centrifuging at 6,000 × g for 20 min at 4°C. If the target protein is secreted into the culture medium, filter the supernatant by passing it through a < 0.45-μm pore size membrane and collect the filtrate. If needed, add appropriate protease inhibitors to the filtrate to prevent proteolytic degradation of secreted target proteins. If the target protein is expressed in the cytoplasm of the host cells, discard the supernatant after centrifugation and retain the cell pellet. 3.2. Uniform 2H, 15 N-Labeling Using P. pastoris

Deuterium isotope-labeling of target proteins is one of the most important techniques for protein NMR studies, especially for analyses of large molecular weight (MW > 25 K) proteins and in crosssaturation experiments to identify intermolecular-binding sites (6, 21–24). The P. pastoris expression system can be used to overexpress deuterium-labeled heterologous proteins. For the efficient production of deuterated target proteins, cells should be adapted to grow in deuterated broth medium. Adaptation is achieved by multistage subculturing in which the deuterium concentration is raised in a stepwise fashion. For instance, subculturing of cells is performed using semideuterated (25–95% 2H2O) medium, with subsequent culturing in fully deuterated medium (14) (see Note 4). In the case of uniform 2H-labeling, using a 2 H-labeled carbon source (such as [2H4]methanol) only during the induction phase is insufficient for producing fully deuterated target proteins (a considerable amount of protons remain on methyl and Lys δ/ε groups) (14). To achieve the nearly-complete deuteration level required for cross-saturation experiments, a deuterium-labeled carbon source, such as D-[2H7]glucose should also be used during the cell growth phase (prior to the induction phase) (7, 14). In this section, we describe a cultivation procedure for overexpressing perdeuterated heterologous proteins in P. pastoris. Using this procedure, Ichikawa and coworkers successfully prepared a discoidin domain of DDR2 that was more than 95% deuterated, and clearly identified the collagen-binding site of DDR2 through transferred cross-saturation experiments (7). 1. Prepare necessary media: (a) Autoclave a 1-L baffled flask and a 2-L fermentation vessel, and dry them in an oven at 60–80°C (for at least 48 h) to completely remove residual H2O. (b) Prepare 1.3 L of 15N-BM(2H2O) medium in a 2-L media bottle, 10 mL of 10% unlabeled D-glucose(2H2O), and 15 mL of 10% D-[2H7]glucose(2H2O) solutions.

2

Isotopic Labeling of Heterologous Proteins in the Yeast…

29

(c) Prepare 5 mL each of 15N-BMD(90% 2H2O), 15N-BMD (2H2O), and 2H, 15N-BMD(2H2O) media in 50-mL Corning tubes. (d) Prepare 300 mL of 2H, 15N-BMD(2H2O) medium in a sterilize, well-dried 1-L baffled flask. (e) Prepare 2H, 15N-BMM(2H2O) medium by mixing 1 L of 15 N-BM(2H2O) medium, 0.5–1.0 mL of antifoaming agent and 5 mL of [2H4]methanol in the sterilize, well-dried 2-L fermentation vessel. 2. Using a sterile loop, scoop a small aliquot of P. pastoris transformants from a frozen glycerol stock and streak it onto a YPDS agar plate containing Zeocin™. Incubate the plate for 24–48 h at 30°C. 3. To produce the primary culture, inoculate 5 mL of BMGY medium in a sterile 50-mL Corning tube with a fresh single colony of the P. pastoris transformant from the YPDS agar plate and shake at 200–250 rpm at 30°C for 18–24 h. 4. Add 0.1 mL of the primary culture to 5 mL of fresh, sterile 15 N-BMD(90% 2H2O) medium in a sterile 50-mL Corning tube. Shake the culture tube at 200–300 rpm at 30°C until the cell density reaches an OD600 of approximately 3–5. 5. Add 0.1 mL of the step 4 culture to 5 mL of fresh, sterile 15 N-BMD(2H2O) medium (in a sterile 50-mL Corning tube). Shake the culture tube at 200–300 rpm at 30°C until the cell density reaches an OD600 of 3–5. 6. Add 0.1 mL of the step 5 culture to 5 mL of fresh, sterile 2H, 15 N-BMD(2H2O) medium in a sterile 50-mL Corning tube. Shake the culture tube at 200–300 rpm at 30°C until the cell density reaches an OD600 of 3–5. 7. Pellet the cells by centrifuging at 3,000 × g for 5 min at 20°C and discard the supernatant. Gently resuspend the pellet with 30 mL of fresh, sterile 2H, 15N-BMD(2H2O) medium and pour the resuspended cells into the remaining 2H, 15N-BMD(2H2O) medium in the 1-L baffled flask to a final volume of 300 mL. Shake the flask at > 200 rpm at 30°C until the cell density reaches an OD600 of 3–4. 8. Pellet the cells in sterile tubes by centrifuging at 2,000 × g for 10 min at 20°C and discard the supernatant. Gently resuspend the pellet with 30 mL of fresh, sterile 2H, 15N-BMM(2H2O) medium and pour the resuspended cells into the remaining sterile 2H, 15N-BMM(2H2O) medium in the 2-L fermentation vessel. The starting OD600 of the culture in the fermenter vessel should be approximately 1 (see Notes 6–8). 9. Assemble the fermentation system Subheading 3.1, step 6 (see Note 13).

as

described

in

30

T. Sugiki et al.

10. Agitate the culture medium at 300–800 rpm with 0.1–0.3 L/min of feeding air at 30°C (see Notes 9 and 10). Add [2H4] methanol (0.5 % (w/v) of the total volume) to the culture medium every 24 h during this induction phase (see Notes 11 and 12). 11. Collect the target proteins as described in Subheading 3.1, step 8. 3.3. Uniform 13C, 15 N-Labeling Using K. lactis

1. Prepare 0.5 L of sterile 13C, 15N-BMD and 1.0 L of 13C, 15N-BMD feeding medium in a 2-L fermentation vessel and 1-L media bottle, respectively. 2. Using a sterile loop, scoop a small aliquot of K. lactis transformants from a frozen glycerol stock and streak it onto a YCBacetamide agar plate. Incubate the plate for 24–48 h at 30°C. 3. To produce the primary culture, inoculate a fresh, single colony of K. lactis transformants into 5 mL of YPD medium (in a 50-mL Corning tube) and shake at 200–250 rpm at 30°C for 48 h to obtain a saturated biomass (OD600 > 20–30). 4. Assemble the fermentation system as described in Subheading 3.1, step 6. Connect the media bottle containing 1 L of fresh 13C, 15N-BMD feeding medium to the inlet port of the vessel with an appropriate length of feeding tube, and attach a peristaltic pump at the midpoint of the feeding tube (Fig. 1). 5. Pellet the primary culture cells by centrifuging at 3,000 × g for 5 min at 20°C and discard the supernatant. Gently resuspend the pellet with 30 mL of fresh, sterile 13C, 15N-BMD medium from the 2-L fermentation vessel, and pour the resuspended cells into the remaining 13C, 15N-BMD medium in the fermentation vessel. Agitate the culture medium at 600–800 rpm with 0.1–0.3 L/min feeding air at 30°C (see Notes 9, 10, and 12). 6. During the fermentation, continuously feed fresh, sterile 13C, 15 N-BMD feeding medium into the fermentation vessel at a constant flow rate of 8.3 mL/h using the peristaltic pump (see Note 14). 7. Pellet the cells by centrifuging at 10,000 × g for 15 min at 4°C. If the target protein is secreted into the culture medium, filter the supernatant by passing it through a < 0.45-μm pore size membrane and collect the filtrate. If needed, add appropriate protease inhibitors to the collected filtrate to prevent proteolytic degradation of the target protein. If the target protein is expressed in the cytoplasm of the host cells, discard the supernatant after centrifugation, and retain the cell pellet.

3.4. Uniform 2H, 15 N-Labeling Using K. lactis

In our experience, perdeuteration (approximately 90% deuteration estimated by MS analysis) of maltose-binding protein (MBP) is successfully achieved using K. lactis with fed-batch fermentation.

2

Isotopic Labeling of Heterologous Proteins in the Yeast…

31

Fig. 1. Overview of the fed-batch fermentation system. (a) Schematic diagram of fed-batch fermentation. (b) Photograph of the assembled fed-batch fermentation device. a: fresh media which is continuously fed into the fermentation vessel, b: peristaltic pump, c: fermentation vessel, d: air flow inlet, and e: air exhaust port.

Furthermore, insertion of one or two subcultivation steps following the primary culture improves cell growth and expression of MBP with a highly efficient (> 92%) level of deuteration (Miyazawa-Onami and coworkers, unpublished data). 1. Prepare necessary media: (a) Autoclave an empty 1-L media bottle and a 2-L fermentation vessel, and dry them in an oven at 60–80°C (for at least 48 h) to completely remove residual H2O. (b) Prepare 0.5 L of fresh, sterile 2H, 15N-BMD(2H2O) and 1.0 L of 2H, 15N-BMD(2H2O) feeding medium in the sterile 2-L fermentation vessel and 1-L media bottle, respectively. 2. Using a sterile loop, scoop a small aliquot of K. lactis transformants from a frozen glycerol stock and streak it onto a YCBacetamide agar plate. Incubate the plate for 24–48 h at 30°C.

32

T. Sugiki et al.

3. To produce the primary culture, inoculate 5 mL of YPD medium in a 50-mL Corning tube using a fresh single colony of the K. lactis transformants from the YCB agar plate and shake at 200–250 rpm at 30°C for 48 h to obtain a saturated biomass (OD600 > 20–30). 4. Assemble the fermentation system as described in Subheading 3.1, step 6 (see Note 13). Connect the bottle containing 1 L of fresh 2H, 15N-BMD(2H2O) feeding medium to the inlet port of the vessel with an appropriate length of feeding tube, and attach a peristaltic pump at the midpoint of the feeding tube (Fig. 1). 5. Pellet the cells by centrifuging at 3,000 × g for 5 min at 20°C and discard the supernatant. Gently resuspend the pellet with 30 mL of fresh, sterile 2H, 15N-BMD(2H2O) medium from the 2-L fermentation vessel, and pour the resuspended cells into the remaining 2H, 15N-BMD(2H2O) medium in the fermentation vessel. Agitate the culture medium at 600–800 rpm with 0.1–0.3 L/min feeding air at 30°C (see Notes 9, 10, and 12). 6. During the fermentation, continuously feed fresh, 2H, 15 N-BMD(2H2O) feeding medium from the 1-L media bottle into the fermentation vessel at a constant flow rate of 8.3 mL/h using the peristaltic pump (see Note 14). 7. Collect the target proteins as described in Subheading 3.3, step 7.

4. Notes 1. Detailed information about the composition of YNB without amino acids and ammonium sulfate can be found in the Becton Dickinson Difco Yeast Media Recipes, available at http://www. bd.com/ds/technicalCenter/inserts/Yeast_Media.pdf. 2. Ammonium sulfate or ammonium chloride is used as a nitrogen source by the yeast. In many cases, 5–10 g/L of ammonium sulfate or ammonium chloride are used in the culture of yeast. In our experience, a sufficient amount of isotope-labeled protein could be expressed while keeping costs to a minimum by using 2 g/L of 15N-ammonium chloride (7). 3. Foaming of the culture medium caused by agitation and aeration will severely affect the yield of target protein due to a reduction in the level of protein expression and partial denaturation of the expressed proteins (25). Foaming should be controlled

2

Isotopic Labeling of Heterologous Proteins in the Yeast…

33

by adding antifoaming agents to the culture medium. We use 0.05–0.10% (w/v) of “Antifoam 204” (supplied from Sigma). 4. For many organisms, 2H2O-containing medium negatively affect physiological processes. In many cases, stagnation of cell growth occurs in 75–100% 2H2O, necessitating adaptation to the deuterated medium by subcultivating the cells several times prior to the induction of protein expression (2). Furthermore, the selection of transformants using deuterated agar plates may be effective in generating special transformants that are more adaptable to deuterated medium. 5. Although glycerol is widely used as a carbon source of P. pastoris during growth phase, in the case of 13C-labeling, 13C-glycerol can be replaced by 13C-glucose to reduce labeling costs (9, 26, 27). 6. An OD600 = 1 is equivalent to 5 × 107 P. pastoris cells (2). 7. Nonmethanolic carbon sources, especially glucose, should be completely eliminated (or consumed) prior to the addition of methanol to achieve full induction efficiency of the AOX1 promoter (2, 7, 20). However, the use of mixtures of carbon sources during the induction phase to improve cell growth and the production of target proteins has been reported (28–32). 8. The optimal starting point of the induction phase should be determined by performing a time course study of cell growth in a “cold-run” prior to beginning a “hot-run,” since the optimal point for starting induction varies according to the type of target protein and culture conditions. 9. The level of oxygen supplied to the fermentation medium should be kept at 0.1–0.3 vvm (volume of oxygen (liters) per volume of fermentation culture (liters) per minute) during the cultivation. This level can be easily achieved using any glass fermenter and mixing the broth medium via an impeller in an air feeding condition. Maintaining an adequate concentration of dissolved O2 during cultivation is crucially important for sufficient cell growth and protein expression (2, 20, 33). 10. Proteolytic degradation of heterologous proteins is one of the drawbacks of the secretory expression strategy. In our experience, this often occurs in a culture grown in a poor-nutrient medium such as the one that may be found under isotope labeling conditions. To prevent extensive proteolytic degradation of target proteins, the following measures can be taken (1) optimizing the pH range of the culture medium (34); (2) cultivating at low-temperature (35–38); (3) adding extracts of algae, such as BioExpress. (26); (4) adding protease inhibitors (2, 27, 39); however, some types of protease inhibitors are toxic; and (5) utilizing protease-deficient strains (34, 40). Several

34

T. Sugiki et al.

protease-deficient strains of P. pastoris and K. lactis are commercially available. 11. In the P. pastoris expression system, the methanol concentration during induction directly affects cell growth and protein production since an excess of methanol (> 1–2% (w/v)) and an excessive rate of feeding is toxic to cells (5). Monitoring the level of dissolved O2 in the culture medium is a convenient method for assessing the timing for feeding and the appropriate dose of methanol because sharp “spike” signals are detected by the O2 electrode when the carbon sources are completely consumed and/or when the concentration of methanol reaches a toxic level (20, 33). 12. The typical induction time is between 48 and 96 h. However, in the case of a deuterium-labeling culture, an induction period longer than 96 h is typically required. The optimal induction period and the amount of methanol should be determined by following the time course of target protein expression level using SDS-PAGE. 13. To achieve a higher level of deuteration, a glass tube filled with dried granular calcium chloride should be connected in front of the air inlet of the fermentation vessel to remove H2O from the supplied air. 14. The optimal culture conditions (composition of media, pH, temperature, etc.) vary according to the type of host and target proteins. In our experience, the most critical factors for obtaining sufficient protein expression using K. lactis are the concentration of the carbon source and the concentration of dissolved oxygen. Especially, in cases involving the use of minimal media for isotope labeling, we recommend fermentation cultivation rather than flask culture. Furthermore, addition of isotopically labeled extracts of algae, such as Celtone, BioExpress (both supplied by Cambridge Isotope Laboratories, Inc.) or C.H.L. (Chlorella Industry Co., Ltd.) significantly improves cell growth and protein expression (41).

Acknowledgments This work was financially supported in part by the Ministry of Economy, Trade and Industry (METI) and the New Energy and Industrial Technology Development Organization (NEDO).

2

Isotopic Labeling of Heterologous Proteins in the Yeast…

35

References 1. Cregg, J. M., Vedvick, T. S., and Raschke, W. C. (1993) Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology 11, 905–910. 2. Pickford, A. R., and O’Leary, J. M. (2008) Isotopic labeling of recombinant proteins from the methylotrophic yeast Pichia pastoris, in Methods in Molecular Biology (Downing, A. K., Ed.), vol. 278, pp. 17–33, Humana Press, Totowa, NJ. 3. Lin Cereghino, G. P., Cereghino, J. L., Ilgen, C., and Cregg, J. M. (2002) Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr. Opin. Biotechnol. 13, 329–332. 4. Daley, R., and Hearn, M. T. (2005) Expression of heterologous proteins in Pichia pastoris : a useful experimental tool in protein engineering and production. J. Mol. Recognit. 18, 119–138. 5. Cos, O., Ramόn, R., Montesinos, J. L., and Valero F. (2006) Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review. Microbial Cell Factories 5, 17–36. 6. Takahashi, H., and Shimada, I. (2010) Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic cells. J. Biomol. NMR 46, 3–10. 7. Invitrogen Corporation. EasySelect™ Pichia Expression Kit: A manual of methods for expression of recombinant proteins using pPICZ and pPICZα in Pichia pastoris, Version I. Available at http://tools.invitrogen.com/content/sfs/manuals/easyselect_man.pdf. 8. Laroche, Y., Strome, V., De Meutter, J., Messens, J., and Lauwereys, M. (1994) Highlevel secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast, Pichia pastoris. Bio/Technol 12, 1119–1124. 9. Denton, H., Smith, M., Husi, H., Uhrin, D., Barlow, P. N., Batt, C. A. , and Sawyer, L. (1998) Isotopically labeled bovine β-lactoglobulin for NMR studies expressed in Pichia pastoris. Protein Expr. Purif. 14, 97–103. 10. Wood, M. J., and Komives, E. A. (1999) Production of large quantities of isotopically labeled protein in Pichia pastoris by fermentation.. J. Biomol. NMR 13, 149–159. 11. Mine, S., Ueda, T., Hashimoto, Y., Tanaka, Y., and Imoto, T. (1999) High-level expression of uniformly 15N-labeled hen lysozyme in Pichia pastoris and identification of the site in hen

lysozyme where phosphate ion binds using NMR measurements. FEBS Lett. 448, 33–37. 12. van den Burg, H. A., de Wit, P. J., and Vervoort, J. (2001) Efficient 13C/15N double labeling of the avirulence protein AVR4 in a methanolutilizing strain (Mut+) of Pichia pastoris. J. Biomol. NMR 20, 251–261. 13. Rodriguez, E., and Krishna, N. R. (2001) An economical method for 13C/15N isotopic labeling of proteins expressed in Pichia pastoris. J. Biochem. 130, 19–22. 14. Morgan, W. D., Kragt, A., and Feeney, J. (2000) Expression of deuterium-isotope-labelled protein in the yeast Pichia pastoris for NMR study. J. Biomol. NMR 17, 337–347. 15. Ichikawa, M., Osawa, M., Nishida, N., Goshima, N., Nomura, N., and Shimada, I. (2007) Structural basis of the collagen-binding mode of discoidin domain receptor 2, EMBO J. 26, 4168–4176. 16. New England Biolabs, Inc. K. lactis Protein Expression Kit: Instruction manual. Available at http:/ /www.neb.com/nebecomm/ManualFiles/ manualE1000.pdf. 17. Colussi, P. A., and Taron, C. H. (2005) Kluyveromyces lactis LAC4 promoter variants that lack function in bacteria but retain full function in K. lactis, Appl. Environ. Microbiol. 71, 7092–7098. 18. Read, J. D., Colussi, P. A., Ganatra, M. B., and Taron, C. H. (2007) Acetamide selection of Kluyveromyces lactis cells transformed with an integrative vector leads to high-frequency formation of multicopy strains. Appl. Environ. Microbiol. 73, 5088–5096. 19. Sugiki, T., Shimada, I., and Takahashi, H. (2008) Stable isotope labeling of protein by Kluyveromyces lactis for NMR study. J. Biomol. NMR 42, 159–162. 20. Invitrogen Corporation. Pichia Fermentation Process Guidelines. Available at http://toolsja. invitrogen.com/content/sfs/manuals/pichiaferm_prot.pdf. 21. Takahashi, H., Nakanishi, T., Kami, K., Arata, Y., and Shimada, I. (2000) A novel NMR method for determining the interface of large protein-protein complexes. Nat. Struct. Biol. 7, 220–223. 22. Nakanishi, T., Miyazawa, M., Sakakura, M., Terasawa, H., Takahashi, H., and Shimada, I. (2002) Determination of the interface of a large protein complex by transferred cross-saturation measurements. J. Mol. Biol. 318, 245–249. 23. Takahashi, H., Miyazawa, M., Ina, Y., Fukunishi, Y., Mizukoshi, Y., Nakamura, H., and Shimada,

36

T. Sugiki et al.

I. (2006) Utilization of methyl proton resonances in cross-saturation measurement for determining the interfaces of large protein-protein complexes. J. Biomol. NMR 34, 167–177. 24. Shimada, I., Ueda, T., Matsumoto, M., Sakakura, M., Osawa, M., Takeuchi, K., Nishida, N., and Takahashi, H. (2008) Cross-saturation and transferred cross-saturation experiments. Prog. Nucl. Magn. Reson. Spectrosc. 54, 123–140. 25. Koch, V., Rüffer, H.-M., Schügerl, K., Innertsberger, E., Menzel, H., and Weis, J. (1995) Effect of antifoam agents on the medium and microbial cell properties and process performance in small and large reactors. Process Biochem. 30, 435–446. 26. Macauley-Patrick, S., Fazenda, M. L., McNeil, B., and Harvey, L. M. (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22, 249–270. 27. Shapiro, R. I., Wen, D., Levesque, M., Hronowski, X., Gill, A., Garber, E. A., Galdes, A., Strauch, K. L., and Taylor, F. R. (2003) Expression of sonic hedgehog-Fc fusion protein in Pichia pastoris. Identification and control of post-translational, chemical, and proteolytic modifications. Protein Expr. Purif. 29, 272–283. 28. Files, D., Ogawa, M., Scaman, C. H., and Baldwin, S. A. (2001) A Pichia pastoris fermentation process for producing high-levels of recombinant human cystatin-C. Enzyme Microbial Technol. 29, 335–340. 29. Zhang, W., Hywood Potter K. J., Plantz, B. A., Schlegel, V. L., Smith, L. A., and Meagher, M. M. (2003) Pichia pastoris fermentation with mixed-feeds of glycerol and methanol: growth kinetics and production improvement. J. Ind. Microbiol. Biotechnol. 30, 210–215. 30. Xie, J., Zhang, L., Ye, Q., Zhou, Q., Xin, L., Du, P., and Gan, R. (2003) Angiostatin production in cultivation of recombinant Pichia pastoris fed with mixed carbon sources. Biotechnol. Lett. 25, 173–177. 31. McGrew, J. T., Leiske, D., Dell, B., Klinke, R., Krasts, D., Wee, S. F., Abbott, N., Armitage, R., and Harrington, K. (1997) Expression of trimeric CD40 ligand in Pichia pastoris: use of a rapid method to detect high-level expressing transformants. Gene 187, 193–200. 32. d’Anjou, M. C., and Daugulis, A. J. (2001) A rational approach to improving productivity in

Pichia pastoris fermentation. Biotechnol. Bioeng 72, 1–11. 33. Cai, M., Huang, Y., Sakaguchi, K., Clore, G. M., Gronenborn, A. M., and Craigie, R. (1998) An efficient and cost-effective isotope labeling protocol for proteins expressed in Escherichia coli. J. Biomol. NMR 11, 97–102. 34. Zhang, Y., Liu, R., and Wu, X. (2007) The proteolytic systems and heterologous proteins degradation in the methylotrophic yeast Pichia pastoris. Annal. Microbiol. 57, 553–560. 35. Li, Z., Xiong, F., Lin, Q., d’Anjou, M., Daugulis, A. J., Yang, D. S., and Hew, C. L. (2001) Lowtemperature increases the yield of biological active herring antifreeze protein in Pichia pastoris. Protein Expr. Purif. 21, 438–445. 36. Jahic, M., Gustavsson, M., Jansen, A.-K., Martinelle, M., and Enfors, S.-O. (2003) Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes. J. Biotechnol. 102, 45–53. 37. Jahic, M., Wallberg, F., Bollok, M., Garcia, P., and Enfors, S.-O. (2003) Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures. Microbial Cell Factories 2, 6–17. 38. Surribas, A., Stahn, R., Montesinos, J. L., Enfors, S.-O., Valero, F., and Jahic, M. (2007) Production of Rhizopus oryzae lipase from Pichia pastoris using alternative operational strategies. J. Biotechnol. 130, 291–299. 39. Shi, X., Karkut T., Chamankhah, M., AltingMees, M., Hemmingsen, S. M., and Hegedus, D. (2003) Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris. Protein Expr. Purif. 28, 321–330. 40. Yao, X. Q., Zhao, H. L., Xue, C., Zhang, W., Xiong, X. H., Wang, Z. W., Li, X. Y., and Liu, Z. M. (2009) Degradation of HSAAX15(R13K) when expressed in Pichia pastoris can be reduced via the disruption of YPS1 gene in this yeast. J. Biotechnol. 139, 131–136. 41. Madduri, K., Badger, M., Li, Z.-S., Xu, X., Thornburgh, S., Evans, S., and Dhadialla, T. S. (2009) Development of stable isotope and selenomethionine labeling methods for proteins expressed in Pseudomonas fluorescens. Protein Expr. Purif. 65, 57–65.

Chapter 3 Isotope Labeling in Insect Cells Krishna Saxena, Arpana Dutta, Judith Klein-Seetharaman, and Harald Schwalbe Abstract Recent years have seen remarkable progress in applying nuclear magnetic resonance (NMR) spectroscopy to proteins that have traditionally been difficult to study due to issues with folding, posttranslational modification, and expression levels or combinations thereof. In particular, insect cells have proved useful in allowing large quantities of isotope-labeled, functional proteins to be obtained and purified to homogeneity, allowing study of their structures and dynamics by using NMR. Here, we provide protocols that have proven successful in such endeavors. Key words: Isotope labeling, Baculovirus, Nuclear magnetic resonance, Recombinant protein expression, Insect cells

1. Introduction: Baculovirus-Insect Cell Expression System

Isotope labeling of proteins represents an important and often required tool for the application of nuclear magnetic resonance (NMR) spectroscopy to investigate the structure and dynamics of proteins. So far, the great majority of isotope-labeled proteins have been expressed in Escherichia coli (1) because of the ease of cloning and expressing proteins at low cost. When possible, protein production should be performed in a prokaryotic system (i.e., E. coli), since this strategy is the most cost-effective and allows the most flexibility. However, human or complex proteins often cannot be expressed in E. coli in an active, correctly folded, posttranslationally modified form (glycosylated, phosphorylated, etc.). The capacity of E. coli for protein folding and forming disulfide bonds is not

Alexander Shekhtman and David S. Burz (eds.), Protein NMR Techniques, Methods in Molecular Biology, vol. 831, DOI 10.1007/978-1-61779-480-3_3, © Springer Science+Business Media, LLC 2012

37

38

K. Saxena et al.

sufficient for many recombinant proteins, although there are a number of new developments to overcome these limitations: 1. Decreasing the temperature of the cell culture (2, 3) 2. Coexpressing molecular chaperones (4) 3. Fusing highly soluble tags (gst, mbp, trxa, nusa, sumo, etc.) to the target proteins (5) 4. Overexpressing (6, 7) the target protein or using an engineered E. coli strain capable of forming disulfide bonds in the cytoplasm (e.g., Shuffle, New England Biolabs) 5. Refolding in vitro However, low-expression yield and solubility problems of the target protein in E. coli often force the change from a bacterial recombinant protein expression system to a eukaryotic host. Numerous eukaryotic-based expression systems are currently available in protein expression laboratories. For NMR use, only three expression hosts are generally considered: yeast (Pichia pastoris (8), Hansenula polymorpha or Kluyveromyces lactis), baculovirusmediated insect cells, and mammalian cells. More recently, the generation of baculoviruses harboring mammalian promoters (BacMams) have extended the use of baculovirus-mediated expression systems (BvE) to the development of gene delivery (9) and expression vectors in mammalian cells (10). BacMams cannot replicate in mammalian cells, which renders them a much safer alternative to conventional virus vectors (11). Yeast offers a powerful, simple system for expressing recombinant proteins. Besides the capability of performing posttranslational modification to the recombinant protein, the main advantage of these expression hosts is the feasibility of isotope labeling in simple minimal-defined media. Therefore, the costs of 15N, 13C, or 2 H uniform isotope incorporation are negligible in comparison to the costs of other eukaryotic cell media. Higher eukaryotes need well-defined expression media supplemented with expensive, labeled amino acids. But there are also disadvantages to recombinant protein expression in yeast. Since they provide N- and O-linked high-mannose-type glycans that could be immunogenic in humans (12, 13), the production of glycosylated proteins in yeast is not optimal, especially if glycosylation is required for the biological functionality of the target protein. Additionally, yeast cannot perform tyrosine O-sulfation (14), and proteins whose native forms are nonglycosylated may be hyperglycosylated when expressed in yeast (15). A recent study reported the successful reengineering of the glycosylation pathways in P. pastoris to allow the expression of recombinant proteins with human-type glycans (16). This may allow future improvements in the expression of glycosylated proteins in yeast. However, glycosylation is not the only challenge for successful expression of proteins in yeast. Certain proteins are

3

Isotope Labeling in Insect Cells

39

expressed at low levels even when glycosylation is not necessarily an issue (17–19). While the reasons are not fully clear, low-expression yields have been attributed to defects in the ER folding machinery (20–22). Even for proteins in which high-expression yields can be obtained, much of the protein may be misfolded (23). In order to detect which part of the folding machinery may be responsible, protein disulfide isomerase (PDI), an enzyme that catalyzes disulfide exchange in the ER, was overexpressed and found to enhance protein yields (24, 25). On the other hand, human adenosine A2A receptor levels were not found to increase with an increase in PDI expression (26). Thus, the process of disulfide bond formation in yeast remains uncertain. Finally, the case is most uncertain for membrane proteins, where there is a lack of knowledge on folding mechanisms and also on the way chaperones and the translocon participate in the folding pathway. Therefore, it is currently recommended to use higher eukaryotic hosts with advanced cell machinery systems for the production of recombinant proteins that have to be glycosylated, disulfide bonded, and/or membrane inserted for functional activity. Advanced expression systems with higher cell machineries for posttranslational modifications are offered by the baculovirusmediated expression system in insect cells (BvE) and mammalian expression methods. The BvE is one of the most efficient and popular systems among the eukaryotic hosts to use for expressing recombinant proteins. Therefore, its application is widespread in industrial as well as in academic environments for structural and functional studies of diverse proteins. However, for the production of therapeutic recombinant proteins, mammalian expression systems (mainly Chinese hamster ovary cells (CHO) and Human embryonic kidney (HEK) 293 cells) are required. Since CHO and HEK293 cells have been extensively characterized and have the ability for human-like glycosylation, the production of recombinant therapeutic monoclonal antibodies and Fc fusion proteins in this host is safe. The rate of production of therapeutic proteins, the largest class of new products being developed by the biopharmaceutical industry, has increased significantly in recent years (27). Mammalian expression systems have conventionally been considered to be too weak and inefficient for protein expression. However, recent advances have significantly improved the expression levels of these systems. This chapter and the following one, therefore, attempt to provide an overview of some of the recent developments in expression strategies for baculovirus-mediated insect cell and mammalian expression systems in view of NMR investigations. Since NMR requires isotope-labeled protein, the focus of this article is directed toward strategies to incorporate stable isotopes (15N, 13C) into the target protein in insect and mammalian cells (see Chapter 4). A major bottleneck of uniform labeling in higher eukaryotes is the high cost of complex medium

40

K. Saxena et al.

with labeled amino acids. Another limitation of these hosts is that they cannot survive in deuterium oxide (D2O)-containing media, so cost-effective generation of perdeuterated proteins is not available, either for insect or for mammalian cell systems. It is beyond the scope of both chapters to discuss features of the BvE or mammalian expression systems in detail. Comprehensive guides and detailed methodologies for the construction and analysis of recombinant baculovirus for insect cell expression, maintenance of insect cells in culture, and analysis of recombinant protein expression can be found elsewhere (28, 29), including in Baculovirus Manuals from Invitrogen, Pharmingen, Novagen, and others. However, given the number of different BvE strategies, we begin this chapter by explaining the principle of BvE and subsequently give a short survey of BvE options. 1.1. Principle of Baculovirus-Mediated Recombinant Protein Expression

The insect cell baculovirus-mediated expression system (BvE) is a powerful platform to rapidly produce high levels of recombinant proteins (see ref. 28 for an excellent review). Unlike bacterial expression hosts, the baculovirus system relies on a eukaryotic expression system and thus offers protein modification, sorting, and transportation machineries similar to those found in higher eukaryotic organisms. Baculoviruses are insect viruses that predominantly infect insect larvae of the order Lepidoptera (butterflies and moths) (30). A baculovirus expression vector is a recombinant baculovirus that has been genetically modified to contain a foreign gene of interest, which can be expressed in insect cells under the control of a baculovirus gene promoter. The BvE uses a helper-independent virus that can be propagated to high titers in insect cells adapted for growth in suspension cultures, enabling the production of large amounts of protein with relative ease (31). Finally, baculoviruses are noninfectious to vertebrates and their promoters have been shown to be inactive in mammalian cells (32). The most commonly used baculovirus for recombinant protein expression is Autographa californica, a multicapsid nucleopolyhedrovirus (AcMNPV) (33). AcMNPV is a large (130 kb), lytic, double-stranded DNA virus and can accommodate large segments of foreign DNA for the expression of recombinant protein (34). The BvE is based on the infection of cultured insect cells by a recombinant virus vector in which the target DNA (or multiple genes) is integrated under the control of the strong viral polyhedron promoter (28). The polyhedrin gene (polh) is necessary for the formation of polyhedral or occlusion bodies in the cell nucleus, but is nonessential for viral replication in insect cells. Polyhedra are large particles that appear in the nuclei of AcMNPV-infected insect cells. The first recombinant baculoviruses were generated by replacing the viral polyhedrin gene with a foreign gene of interest through homologous recombination (33). Homologous exchange between the flanking sequences common to both DNA molecules facilitates

3

Isotope Labeling in Insect Cells

41

the insertion of the gene of interest into the viral genome at the polh locus, resulting in the production of a recombinant virus genome and allowing the powerful polyhedron promoter to drive protein expression of the foreign gene. Since the efficiency of homologous recombination is quite low, identification, isolation, and selection of recombinant virus were traditionally achieved by labor-intensive, technically demanding plaque assays. Due to the deletion of the polyhedrin gene, the recombinant plaque has a more clearly distinct morphology than the parental virus containing the polh gene. Subsequently, additional rounds of plaque screening are required to separate the desired recombinant virus from the parental wild-type virus. However, discriminating between polyhedron-positive and -negative plaques and isolating recombinant virus turned out to be a serious problem for many investigators who used the BvE for the production of recombinant proteins. Nowadays, these technical issues and the time-consuming plaque purification processes are eliminated. In the next section, some of the key developments in the BvE are presented and discussed. 1.2. Commercially Available Baculovirus Expression Systems

Generally, the baculovirus genome is considered too large to insert a foreign gene directly. In most applications of the BvE, the gene of interest is therefore cloned into a transfer vector, which contains sequences that flank the polyhedron gene in the baculovirus genome. The virus genome and the transfer vector are cotransfected into the insect cells and the gene of interest inserts into the virus genome via homologous recombination (see Fig. 1) under the control of the strong late viral polyhedrin promoter (35). Since a mixture of recombinant and original parental virus is produced after the initial replication, time-consuming plaque purification and isolation are required before protein expression can proceed. BacVector (Merck Biosciences), Baculo-Gold and pBacPAK (BD Biosciences), and Bac-N-Blue (Invitrogen) are commercially available BvEs that use homologous recombination to integrate foreign genes into the virus genome. New developments in generating recombinant virus by using site-specific transpositions (Bac-to-Bac or BaculoDirect, Invitrogen) or progress in recombination methodology with an engineered baculovirus containing a lethal mutation in an essential gene (open reading frame (ORF1629), flashBAC from Oxford Expression Technologies, or BacMagic from EMD Chemicals, Novagen) have facilitated the use of BvE for a larger user community (28). Generally, these improvement strategies can be classified into transfer plasmid modifications and parental baculovirus genome modification (28). The flashBAC and BacMagic are the most promising BvEs so far, since the efficiency of recombination in both systems is 100%. Therefore, these BvEs overcome the requirement of timeconsuming plaque assays and protein expression can be started directly after one or two rounds of virus amplification. This technology

42

K. Saxena et al.

Fig. 1. Construction of baculovirus recombinants with Novagen® BacMagic™ system. This expression system is based on a modified baculovirus genome containing a bacterial artificial chromosome (BAC) at the polyhedrin locus and a partial deletion of the essential ORF1629 viral gene. The BacMagic DNA is mixed with a transfer vector, containing a foreign gene at the polh locus and the complete ORF1629, to generate the recombinant virus via homologous recombination in insect cells. Picture modified from the Novagen manual. © EMD Chemicals Inc., an Affiliate of Merck KGaA. Darmstadt, Germany. BacMagic™ and Novagen® are trademarks of Merck KGaA.

reduces the production of recombinant virus to a one-step procedure, fully amenable to high-throughput and automated production systems. Moreover, this approach is compatible with all baculovirus transfer vectors based on homologous recombination in insect cells at the polyhedrin locus, including several multigene coexpression plasmids. The technology of the flashBAC and BacMagic is driven by a modified bacmid, in which the baculovirus genome AcMNPV with a portion of the essential viral gene (ORF1629) deleted. In addition, a bacterial artificial chromosome (BAC) replaces the polyhedrin-coding region. This combination prevents nonrecombinant virus from replicating in insect cells, yet allows the viral DNA to be propagated as circular DNA in bacteria. This circular viral DNA is then isolated and purified from bacterial cells (flashBAC or BacMagic DNA provided in the kits). Homologous recombination with a compatible expression plasmid (containing the gene of interest

3

Isotope Labeling in Insect Cells

43

flanked by the lef2 and ORF1629 recombination sites) restores the function of the viral ORF1629 allowing the virus DNA to replicate and replaces the BAC sequence with the target coding sequence under the control of the polyhedrin promoter (Fig. 1). Since only recombinant viruses with a restored ORF1629 can replicate, this results in a unique recombinant virus population. This population can then be used directly to infect a larger insect cell culture (50–200 mL) to produce a high-titer working stock. 1.3. Insect Cell Lines

The main insect cell lines used for cotransfections and baculovirus amplification are Spodoptera frugiperda Sf9 or Sf21 (derivatives of the fall armyworm). Trichoplusia ni BTI 5B1-4 (36) (High Five™) cells are generally used for the production of secreted recombinant proteins and not for virus production because of the increased possibility of generating virus mutants (37). Due to the high-mannose and paucimannose types of glycosylation that are obtained in insect cells, no therapeutic protein is currently produced using this system as this would compromise in vivo bioactivity and potentially induce allergenic reactions. Engineering insect cells with glycosyltransferases allows the production of proteins with mammaliantype sugars (38).

1.4. Expression of Labeled Recombinant Protein in the Baculovirus Expression System

Due to the high costs of incorporating stable isotopes into insect cells, it is recommended that the recombinant protein expression is optimized using the chosen BvE before starting with labeled fermentation. There are only a few studies reporting the incorporation of stable isotopes into proteins expressed by baculovirusmediated insect cell expression (39–47). In contrast to the initial trials of amino acid-type selective labeling of proteins in user-defined insect cell media, nowadays there are commercial media (BioExpress-2000, Cambridge Isotope Laboratories, CIL) available for the different labeling applications. Expression of uniformly 13C- 15N-labeled Abelson Kinase domain (13C–15N BioExpress-2000) was the first example of backbone NMR resonance assignments of a recombinant protein expressed using the BvE (48). So far, most uniform labeling protein work in insect cells is only performed by industrial research groups due to the extraordinary costs of the required media. It is not possible to cultivate insect cells in minimal medium, since this host requires essential amino acids for its growth. Reports of selective amino acid isotope labeling in BvE are more frequently cited in the literature, since this approach is easy and fast and does not require expensive medium for labeling. Even in the absence of a backbone assignment of the target protein, structural information can be deduced from the selective labeling approach based on an existing X-ray structure of the protein. From a practical aspect, it should be considered that there are essential and nonessential amino acids (alanine, cysteine, glutamic acid, glutamine, aspartic acid, and asparagine) in insect cells whose content in the medium

44

K. Saxena et al. NH4

Glutamin

Lysine

Aspartate

Asparagine

Glutamate

Tyrosine

Alanine

Phenylalanine

Valine

Leucine

Isoleucine

Glycine

Serine

Fig. 2. A schematic presentation of amino acid metabolism in E. coli and Sf9 with respect to 15N: The black arrows symbolize pathways present in both expression hosts. Pathways that only exist in E. coli are shown in grey. The strength of the arrows reflects the intensity of the conversion. Picture modified from Bruggert et al. (42).

depends on the specific provider of the medium. Before starting with site-specific amino acid labeling, the unlabeled quantity of the desired amino acid in the medium should be checked to calculate the required amount of the amino acid to be labeled. In Fig. 2, a schematic presentation of amino acid metabolism is shown for E. coli and insect cells (42). Interestingly, selective labeling of amino acids in insect cells is more effective than in bacteria, since the amino acid pathways in insect cells do not harbor as many aminotransferases as in prokaryotes, which leads to cross-labeling problems.

2. Materials 2.1. Cotransfection of Insect Cells

1. 35-mm2 tissue culture dishes. 2. Sf9 or Sf21 insect cells. 3. SF900 II insect cell culture medium (serum-free, antibioticfree; Invitrogen). 4. Insect GeneJuice® transfection reagent (Novagen) (see Note 1). 5. BacMagic DNA: 100 ng (5 μL) per cotransfection (20 ng/μL). 6. Sterile baculovirus transfer vector DNA containing the gene under investigation (500 ng per cotransfection). 7. Plastic box to house dishes in the incubator. 8. Sterile pipettes, bijoux (sterile tubes).

3

2.2. Amplification of Recombinant Virus

Isotope Labeling in Insect Cells

45

1. Recombinant virus seed stock (Subheading 3.1). 2. Sf 9 insect cells. 3. SF900 II insect cell culture medium (Subheading 2.1). 4. Inverted phase-contrast microscope.

2.3. Analysis of Recombinant Protein Expression

1. 35-mm2 tissue culture dishes. 2. Sf 9 or High Five insect cells. 3. SF900 II insect cell culture medium (Subheading 2.1). 4. Recombinant virus stock (Subheading 3.2). 5. Phosphate-buffered saline (PBS; Invitrogen): pH 6.2, sterilize by autoclaving.

2.4. Production of Isotopically Labeled Protein

1. Sf 9 insect cells. 2. SF900 II insect cell culture medium (Subheading 2.1). 3. BioExpress-2000-U (CIL): Unlabeled insect cell culture medium. 4. BioExpress-2000-CN (CIL): 15N-, 13C-labeled insect cell culture medium. 5. Recombinant virus stock (Subheading 3.2). 6. Protease inhibitor (Complete™, Roche). 7. PBS (Subheading 2.3).

3. Methods For a general overview of the BvEs and cloning, expression, analysis, and purification of recombinant proteins in insect cells, please refer to ref. 29. The following procedures describe the cloning of a recombinant baculovirus expression vector, production (Subheading 3.1) and amplification of recombinant baculovirus (Subheading 3.2), analysis of recombinant protein expression (Subheading 3.3), and finally production of isotope-labeled protein (Subheading 3.4) expressed in insect cells. All of the procedures are based on the flashBAC or BacMagic systems (no plaque purification required) and must be carried out using sterile technique. Since the flashBAC system is compatible with all transfer vectors designed for homologous recombination in insect cells at the polh l (BacPAK technology), the target gene can be cloned into many suitable transfer vectors (see Note 2). Moreover, this BvE is compatible with traditional (T4 DNA ligase) and elegant recombinatorial cloning techniques, such as Creator or In-Fusion (BD Biosciences) (see Note 3). While Gateway (Invitrogen) is one of the most popular recombinatorial cloning systems, it is limited to

46

K. Saxena et al.

specifically engineered expression vectors with specifically engineered expression vectors with λ recombination sites. These lead to incorporation of additional amino acids into the protein of interest. A new development to avoid multiple cloning of target genes into host-specific expression vectors is triple host transfer vector, pTriEx, from Novagen. Due to the parallel existence of three promoters in this vector series, recombinant protein expression is enabled in vertebrates, insect cells, and bacteria. 3.1. Cotransfection of Insect Cells

For efficient transfection, high-quality DNA of the transfer plasmid is prepared using commercially available plasmid DNA purification kits (Qiagen, Novagen) (see Note 4). Additionally, it is recommended to use fresh, rapidly proliferating cells (see Note 5) for transfection experiments and to have positive and negative transfection controls (see Note 6). 1. For each cotransfection, prepare one 35-mm2 plate. Seed the dishes with insect cells at least 1 h before use. Use 1 × 106 cells/ dish for Sf9 cells and 1.5 × 106 cells/dish for Sf21 cells in 2 mL of SF900 II insect cell culture medium. Allow the cells to attach by incubating at 28°C for 20 min. 2. During the 1-h incubation period, prepare a DNA–liposome complex cotransfection mix of BacMagic DNA and Insect GeneJuice® transfection reagent for each transfection. Assemble the following components, in the order listed, in a sterile tube (bijoux) (see Note 7): 1 mL serum-free, antibiotic-free SF900 II insect cell culture medium 5 μL Insect GeneJuice 5 μL BacMagic DNA (100 ng total) 5 μL Transfer vector DNA (500 ng total) 1.015 mL Total volume 3. Incubate at room temperature for 15–30 min to allow the DNA–liposome complexes to form. 4. Remove the culture medium from the 35-mm2 dishes of cells using a sterile pipette, ensuring that the cell monolayer is not disrupted (see Note 8). 5. Immediately after the medium has been removed from the cells, add the 1 mL of the DNA–liposome complex dropwise to the center of each dish (see Note 9) and incubate in a plastic sandwich box at 28°C for a minimum of 5 h or overnight. 6. After the incubation period, add another 1 mL of SF900 II insect cell culture medium to each dish and continue the incubation for 5 days in total.

3

Isotope Labeling in Insect Cells

47

7. Following the 5-day incubation period (see Note 10), harvest the medium containing the recombinant virus into a sterile bijoux and store in the dark at 4°C. This is the seed stock of recombinant baculovirus (see Note 11). Due to the limited size of the stock, the next step is to amplify the virus recombination sites. 3.2. Amplification of Recombinant Virus

Amplification of the recombinant virus (produced in Subheading 3.1) is necessary before proceeding with recombinant protein expression. The following provides a protocol (adapted from the Novagen and flashBAC Manual) for amplification and preparation of high-titer recombinant virus (passage 1 stock) in cells grown in suspension culture. 1. Observe the health and viability of cells under an inverted phase-contrast microscope (see Note 12). 2. Prepare a 100–200 mL culture of Sf9 cells at an appropriate cell density in serum-free SF900 II insect cell culture medium (e.g., 2 × 106 Sf9 cells/mL in log-phase growth; high aeration is recommended (see Note 13)). Cells should be infected at a multiplicity of infection (MOI) of <1 pfu/cell. 3. Add 0.5 mL of recombinant virus seed stock to the cell culture. Incubate with shaking until the cells are well-infected (usually, 4–5 days) (see Note 14). 4. When the cells appear to be well-infected with virus, harvest the cell culture medium by centrifugation at 1,500 × g for 15 min at 4°C. Remove the supernatant aseptically. Store the supernatant (recombinant virus stock) in the dark at 4°C (see Note 15).

3.3. Analysis of Recombinant Protein Expression

Before proceeding with use of the virus in subsequent protein expression experiments, a plaque assay to determine the accurate titer is strongly recommended by the manufacturer of the flashBAC and BacMagic technology (see Note 16). This allows the calculation of MOI and ensures cross-referencing and reproducibility in the following experiments. However, for analysis of protein expression, it is not absolutely required to know the titer of the recombinant virus (see Note 17). Pilot expression analysis can also be performed by infecting cells with different amounts of P1 virus and monitoring the expression of the recombinant protein. According to the protein expression literature, it is recommended that exponentially growing cells should be infected at a high MOI to ensure that all cells are infected simultaneously and that the culture is synchronous. However, in our lab, various P1 viruses are directly added (0.5–10%) to the insect cells for determining the best ratio for optimal recombinant protein expression without knowing the exact titer of the different viruses. The best time to

48

K. Saxena et al.

harvest the recombinant protein can be examined by taking samples at different time points after infection (see Note 18). It is recommended to also prepare noninfected or mock-infected cells as a control for host cell proteins and a positive control of a successfully tested recombinant virus. Recombinant protein expression can then be evaluated by SDS-PAGE or Western blot analysis. Additionally, protein expression should be compared between Sf9 and High Five cell lines, especially if the protein of interest is secreted (see Note 19). The following protocol is adapted from the Novagen and flashBAC Manual. 1. Seed 35-mm2 dishes (one per virus to be tested plus a positive and negative control) with 1 × 106 of S9 or High Five cells per dish and allow the cells to attach by incubating at 28°C for 1 h. 2. Remove the medium, add 200 μL of the recombinant virus stock to be tested, and incubate at room temperature for an additional hour. 3. Remove the virus inoculum, add 1.5 mL of SF900 II insect cell culture medium, and incubate at 28°C for 48 h. 4. Use a sterile pipette tip to scrape the cells from the dishes into suspension and to transfer them into sterile Eppendorf tubes. 5. Centrifuge the cells at 1,500 × g for 10 min at room temperature. Remove the supernatant and discard (see Note 20). 6. Resuspend the pellet (protein of interest in the cytosol) in 80 μL of PBS. 7. Analyze the extent of protein overexpression by using SDSPAGE and Western blotting if a suitable antibody is available for the recombinant protein (see Note 21 and Fig. 3).

Fig. 3. Protein overexpression and purification using insect cells. SDS PAGE (left ), Western Blot (right ): Protein samples loaded are BC (before Ni-NTA chromatography), FT (Flowthrough), M (Molecular weight marker), W (Wash), E (Elution).

3

3.4. Production of Isotopically Labeled Protein

Isotope Labeling in Insect Cells

49

While fermenters and bioreactors are now routinely used to produce recombinant proteins in insect cells, good yields can be achieved in shake cultures at a fraction of the cost. Large, disposable shake flasks can be obtained that hold up to 1.5 L of insect cell culture on a shaking platform in a warm room or incubator. The most suitable culture protocol for isotope labeling of a recombinant protein in insect cells involves an initial growth phase for the insect cells in an unlabeled growth/expression medium and a subsequent expression phase, where cells are centrifuged and resuspended into the labeling medium prior to infection with the recombinant baculovirus (see Note 22). This centrifugation step allows much higher expression levels of the labeled protein compared to one with growth and expression in labeling medium without the change in medium. Recently, isotope-labeled expression media for insect cells have become commercially available (BioExpress-2000, CIL), but the source and the concentration of the amino acids in these media are not published. However, for site-specific labeling of amino acids in proteins expressed in insect cells, adding the isotopelabeled amino acid of interest to a standard insect cell medium before infection (see Note 23) also works well and the cost for this labeling strategy is comparable to that when using E. coli. The following protocol is adapted from the Application Note: Efficient uniform isotope labeling of proteins expressed in Baculovirus-infected insect cells using BioExpress® 2000 (Insect cell) medium by André Strauss, Gabriele Fendrich, and Wolfgang Jahnke. Prior to performing isotope labeling of a protein, optimize the culture and BV-infection conditions in unlabeled medium (e.g., BioExpress 2000-U or SF900 II for expression of the protein). 1. Cultivate several 100 mL cultures of Sf9 cells, adapted to growth in serum-free SF900 II medium, in 500-mL Erlenmeyer flasks for 3 days at 27°C with shaking at 90 rpm. 2. Prepare the uniform isotope-labeling medium (see Note 24). 3. When the final cell density of the culture has reached 1.5 × 106 cells/mL (~3 days), sterile centrifuge the cells at 400 × g for 20 min at 20°C. 4. Resuspend the pelleted cells in 100-mL portions in labeled BioExpress® 2000-CN medium and transfer them to fresh 500-mL Erlenmeyer flasks. 5. Add the recombinant virus stock at a titer of 0.5–2 × 108 pfu/mL to an MOI = 1–2, according to optimized conditions. 6. Grow the 100 mL cultures of baculovirus-infected Sf9 cells for 3 days post infection in labeled BioExpress® 2000-CN medium at 27°C, with shaking at 90 rpm (see Note 25). 7. Harvest the cells expressing the labeled recombinant protein by centrifuging at 400 × g for 20 min at 20°C (see Note 26); resuspend the pelleted cells in 20 mL of PBS containing

50

K. Saxena et al.

protease inhibitor mix (see Note 27), followed by a second centrifugation in 50-mL plastic tubes at 400 × g for 20 min at 20°C. Store the pelleted cells at −80°C. 8. Isolate and purify the recombinant protein according to protocols generated for the unlabeled protein.

4. Notes 1. Lipofectin® (Invitrogen), FuGENE 6 (Roche), Tfx-20™ (Promega), and CELLFECTIN® (Invitrogen) have also been successfully tested. 2. Several suitable vectors from different providers are available and are summarized: http://www.expressiontechnologies. com/flashBAC/vectors.asp. 3. Ligation protocols are described in the manuals of the specific cloning technology providers. 4. Plasmid DNA purification protocols are provided by the manufacturers of the purification kits. The DNA must be sterile and must be of a quality suitable for transfection into cells. 5. Healthy cells look bright, round, and refractile, and many should be in the process of dividing into daughter cells. 6. A control transfer vector is supplied with the BacMagic kit and can be used to make recombinant virus; the lacZ-positive infected cells can be stained using X-gal. Add 1 mL of appropriate insect cell culture medium containing 15 μL of 2% (w/v) X-gal in N,N-dimethylformamide and incubate at 28°C. After 5 h, the cells and culture medium appear blue in color, confirming the production of recombinant virus. 7. Plasticware used to prepare the transfection mixture has to be made from polystyrene and not from polypropylene, since the complexes bind to polypropylene. 8. When removing liquid from a dish of cells, tip the dish at a 30–60° angle so that the liquid pools toward one side of the dish. It is important not to allow the cell monolayer to dry out at this point. 9. Adding the mixture dropwise should not disturb the cell monolayer if it is done slowly and gently. 10. Cell monolayers in which recombinant virus has been produced appear very different from mock-transfected control cells under the inverted microscope. Control cells form a confluent monolayer while virus-infected cells do not form a confluent monolayer and appear grainy with enlarged nuclei.

3

Isotope Labeling in Insect Cells

51

11. The expected titer of this initial viral seed stock is generally about 1 × 107 pfu/mL. In contrast to virus-free cells, virusinfected cells appear grainy with enlarged nuclei and do not form a confluent monolayer. 12. It is important that the cells are healthy and in the log phase of growth to ensure that virus replication occurs efficiently to generate high-titer stocks of virus for subsequent use in expression studies suitable for transfection into cells. 13. Virus-infected cells have an increased need for oxygen, and therefore the contents of the flasks should be shaken at quite high speeds to maximize aeration. The surface area-to-volume ratio should also be as large as possible for maximum gas exchange. Do not overfill the flasks. 14. Under a phase-contrast inverted microscope, cells infected with virus appear grainy when compared to healthy cells. The infected cells become uniformly rounded and enlarged, with distinct enlarged nuclei. 15. The virus stock can be stored in the dark at 4°C for 6–12 months, although the titer begins to drop after 3–4 months. Titer the virus before use and reamplify if necessary. The addition of 2–5% serum when using serum-free medium can be helpful in avoiding a drop in titer. Virus may be frozen at −80°C for longer periods of time. Avoid multiple freeze–thaw cycles. 16. Protocols for plaque assays can be found in the manuals of Novagen and Oxford Expression Technologies. 17. After cotransfecting and harvesting the seed stock of virus for further amplification, it is possible to harvest the remaining cells from the dish and prepare these for SDS-PAGE/Western blotting. This gives a quick check for gene expression. 18. The most commonly used times are at 24, 48, 72, and 96 h post infection (hpi). Some proteins may be very stable and accumulate to high levels by 96 hpi, and others may start to degrade and thus need to be harvested much earlier. 19. High Five cell lines (Invitrogen) often increase the yield of secreted recombinant proteins. 20. For secreted protein expression, the supernatant has to be analyzed. 21. Standard molecular biology procedures like SDS-PAGE and Western-blotting are precisely described in Molecular Cloning: A Laboratory Manual) (49). 22. A similar cost-reducing labeling strategy in E. coli was also described for the production of isotope-labeled protein by Marley and coworkers (50).

52

K. Saxena et al.

23. 300–600 mg of 15N-Phe or 15N-Leu (per liter culture medium) was immediately added to the insect cells after being infected with virus. The medium used for the amino acid-selective labeling was 1LSF-900 II serum-free medium from Invitrogen. 24. Medium can be stored, and filter-sterilized for several months at 4°C in the dark without loss of capacity for protein expression. Warm to 28°C before using. 25. The harvesting time of the infected cells depends on the stability of the target protein. 26. For secreted protein expression, the supernatant has to be further purified. 27. Protease inhibitor mix: Roche complete (one tablet dissolved in 200 mL of PBS) References 1. Goto, N. K., and Kay, L. E. (2000) New developments in isotope labeling strategies for protein solution NMR spectroscopy. Curr. Opin. Struct. Biol. 10, 585–592. 2. Schein, C. H. (1991) Optimizing protein folding to the native state in bacteria. Curr Opin. Biotechnol. 2, 746–750. 3. Qing, G., Ma, L. C., Khorchid, A., Swapna, G. V., Mal, T. K., Takayama, M. M., Xia, B., Phadtare, S., Ke, H., Acton, T., Montelione, G. T., Ikura, M., and Inouye, M. (2004) Coldshock induced high-yield protein production in Escherichia coli. Nat. Biotechnol. 22, 877–882. 4. Young, J. C., Agashe, V. R., Siegers, K., and Hartl, F. U. (2004) Pathways of chaperonemediated protein folding in the cytosol. Nat. Rev. Mol. Cell. Biol. 5, 781–791. 5. Esposito, D., and Chatterjee, D. K. (2006) Enhancement of soluble protein expression through the use of fusion tags. Curr. Opin. Biotechnol. 17, 353–358. 6. Andersen, C. L., Matthey-Dupraz, A., Missiakas, D., and Raina, S. (1997) A new Escherichia coli gene, dsbG, encodes a periplasmic protein involved in disulphide bond formation, required for recycling DsbA/DsbB and DsbC redox proteins. Mol. Microbiol. 26, 121–132. 7. Bardwell, J. C. (1994) Building bridges: disulphide bond formation in the cell. Mol. Microbiol. 14, 199–205. 8. Pickford, A. R., and O’Leary, J. M. (2004) Isotopic labeling of recombinant proteins from the methylotrophic yeast Pichia pastoris. Methods Mol. Biol. 278, 17–33.

9. Hu, Y. C. (2008) Baculoviral vectors for gene delivery: a review. Curr. Gene Ther. 8, 54–65. 10. Kost, T. A., Condreay, J. P., Ames, R. S., Rees, S., and Romanos, M. A. (2007) Implementation of BacMam virus gene delivery technology in a drug discovery setting. Drug Discov. Today 12, 396–403. 11. Hitchman, R. B., Possee, R. D., and King, L. A. (2009) Baculovirus expression systems for recombinant protein production in insect cells. Recent Pat. Biotechnol. 3, 46–54. 12. Lam, J. S., Huang, H., and Levitz, S. M. (2007) Effect of differential N-linked and O-linked mannosylation on recognition of fungal antigens by dendritic cells. PLoS One 2, e1009. 13. Dasgupta, S., Navarrete, A. M., Bayry, J., Delignat, S., Wootla, B., Andre, S., Christophe, O., Nascimbeni, M., Jacquemin, M., MartinezPomares, L., Geijtenbeek, T. B., Moris, A., Saint-Remy, J. M., Kazatchkine, M. D., Kaveri, S. V., and Lacroix-Desmazes, S. (2007) A role for exposed mannosylations in presentation of human therapeutic self-proteins to CD4+ T lymphocytes. Proc. Natl. Acad. Sci. U.S.A 104, 8965–8970. 14. Moore, K. L. (2003) The biology and enzymology of protein tyrosine O-sulfation. J. Biol. Chem. 278, 24243–24246. 15. Daly, R., and Hearn, M. T. (2005) Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J. Mol. Recognit. 18, 119–138. 16. Hamilton, S. R., and Gerngross, T. U. (2007) Glycosylation engineering in yeast: the advent

3

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

of fully humanized yeast. Curr. Opin. Biotechnol. 18, 387–392. Chisholm, V., Chen, C. Y., Simpson, N. J., and Hitzeman, R. A. (1990) Molecular and genetic approach to enhancing protein secretion. Methods Enzymol. 185, 471–482. Dorner, A. J., and Kaufman, R. J. (1990) Analysis of synthesis, processing, and secretion of proteins expressed in mammalian cells. Methods Enzymol. 185, 577–596. Moir, D. T. (1989) Yeast mutants with increased secretion efficiency. Biotechnology 13, 215–231. Biemans, R., Thines, D., Rutgers, T., De Wilde, M., and Cabezon, T. (1991) The large surface protein of hepatitis B virus is retained in the yeast endoplasmic reticulum and provokes its unique enlargement. DNA Cell. Biol. 10, 191–200. Gennaro, D. E., Hoffstein, S. T., Marks, G., Ramos, L., Oka, M. S., Reff, M. E., Hart, T. K., and Bugelski, P. J. (1991) Quantitative immunocytochemical staining for recombinant tissue-type plasminogen activator in transfected Chinese hamster ovary cells. Proc. Soc. Exp. Biol. Med. 198, 591–598. Shuster, J. R. (1991) Gene expression in yeast: protein secretion. Curr. Opin. Biotechnol. 2, 685–690. Mollaaghababa, R., Davidson, F. F., Kaiser, C., and Khorana, H. G. (1996) Structure and function in rhodopsin: expression of functional mammalian opsin in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A 93, 11482–11486. Robinson, A. S., Hines, V., and Wittrup, K. D. (1994) Protein disulfide isomerase overexpression increases secretion of foreign proteins in Saccharomyces cerevisiae. Biotechnology (N Y) 12, 381–384. Shusta, E. V., Raines, R. T., Pluckthun, A., and Wittrup, K. D. (1998) Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments. Nat. Biotechnol. 16, 773–777. Butz, J. A., Niebauer, R. T., and Robinson, A. S. (2003) Co-expression of molecular chaperones does not improve the heterologous expression of mammalian G-protein coupled receptor expression in yeast. Biotechnol. Bioeng. 84, 292–304. Durocher, Y., and Butler, M. (2009) Expression systems for therapeutic glycoprotein production. Curr. Opin. Biotechnol. 20, 700–707. Jarvis, D. L. (2009) Baculovirus-insect cell expression systems. Methods Enzymol. 463, 191–222.

Isotope Labeling in Insect Cells

53

29. O´Reilly, D. R., Miller, L., Luckow, V.A. (1992) Baculovirus Expression Vectors - a Laboratory Manual. WH Freeman, New York. 30. van Regenmortel, M. H., Mayo, M. A., Fauquet, C. M., and Maniloff, J. (2000) Virus nomenclature: consensus versus chaos. Arch. Virol. 145, 2227–2232. 31. Hunt, I. (2005) From gene to protein: a review of new and enabling technologies for multiparallel protein expression. Protein Expr. Purif. 40, 1–22. 32. Carbonell, L. F., Klowden, M. J., and Miller, L. K. (1985) Baculovirus-mediated expression of bacterial genes in dipteran and mammalian cells. J. Virol. 56, 153–160. 33. Smith, G. E., Summers, M. D., and Fraser, M. J. (1983) Production of human beta interferon in insect cells infected with a baculovirus expression vector. Molecular Cell. Biol. 3, 2156–2165. 34. Ayres, M. D., Howard, S. C., Kuzio, J., LopezFerber, M., and Possee, R. D. (1994) The complete DNA sequence of Autographa californica nuclear polyhedrosis virus. Virology 202, 586–605. 35. Kitts, P. A., and Possee, R. D. (1993) A method for producing recombinant baculovirus expression vectors at high frequency. Biotechniques 14, 810–817. 36. Kost, T. A., Condreay, J. P., and Jarvis, D. L. (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat. Biotechnol. 23, 567–575. 37. Friesen, P. D., and Nissen, M. S. (1990) Gene organization and transcription of TED, a lepidopteran retrotransposon integrated within the baculovirus genome. Mol. Cell. Biol. 10, 3067–3077. 38. Harrison, R. L., and Jarvis, D. L. (2006) Protein N-glycosylation in the baculovirusinsect cell expression system and engineering of insect cells to produce “mammalianized” recombinant glycoproteins. Adv. Virus Res. 68, 159–191. 39. DeLange, F., Klaassen, C. H., Wallace-Williams, S. E., Bovee-Geurts, P. H., Liu, X. M., DeGrip, W. J., and Rothschild, K. J. (1998) Tyrosine structural changes detected during the photoactivation of rhodopsin. J. Biol. Chem. 273, 23735–23739. 40. Creemers, A. F., Klaassen, C. H., Bovee-Geurts, P. H., Kelle, R., Kragl, U., Raap, J., de Grip, W. J., Lugtenburg, J., and de Groot, H. J. (1999) Solid state 15N NMR evidence for a complex Schiff base counterion in the visual G-proteincoupled receptor rhodopsin. Biochemistry 38, 7195–7199.

54

K. Saxena et al.

41. Bellizzi, J. J., Widom, J., Kemp, C. W., and Clardy, J. (1999) Producing selenomethioninelabeled proteins with a baculovirus expression vector system. Structure 7, R263-267. 42. Bruggert, M., Rehm, T., Shanker, S., Georgescu, J., and Holak, T. A. (2003) A novel medium for expression of proteins selectively labeled with 15N-amino acids in Spodoptera frugiperda (Sf9) insect cells. J. Biomol. NMR 25, 335–348. 43. Strauss, A., Bitsch, F., Cutting, B., Fendrich, G., Graff, P., Liebetanz, J., Zurini, M., and Jahnke, W. (2003) Amino-acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells useful for NMR studies. J. Biomol. NMR 26, 367–372. 44. Strauss, A., Bitsch, F., Fendrich, G., Graff, P., Knecht, R., Meyhack, B., and Jahnke, W. (2005) Efficient uniform isotope labeling of Abl kinase expressed in Baculovirus-infected insect cells. J. Biomol. NMR 31, 343–349. 45. Betz, M., Vogtherr, M., Schieborr, U., Elshorst, B., Grimme, S., Pescatore, B., Langer, T., Saxena, K., and Schwalbe, H. (2008) Chemical Biology of Kinases Studied by NMR Spectroscopy, In Chemical Biology (Prof. Dr. Stuart L. Schreiber, P. D. T. M. K. P. D. G., Ed.), pp 852–890.

46. Stockman, B. J., Kothe, M., Kohls, D., Weibley, L., Connolly, B. J., Sheils, A. L., Cao, Q., Cheng, A. C., Yang, L., Kamath, A. V., Ding, Y. H., and Charlton, M. E. (2009) Identification of allosteric PIF-pocket ligands for PDK1 using NMR-based fragment screening and 1H-15N TROSY experiments. Chem. Biol. Drug Des. 73, 179–188. 47. Jahnke, W., Grotzfeld, R. M., Pelle, X., Strauss, A., Fendrich, G., Cowan-Jacob, S. W., Cotesta, S., Fabbro, D., Furet, P., Mestan, J., and Marzinzik, A. L. (2010) Binding or bending: distinction of allosteric Abl kinase agonists from antagonists by an NMR-based conformational assay. J. Am. Chem. Soc. 132, 7043–7048. 48. Vajpai, N., Strauss, A., Fendrich, G., CowanJacob, S. W., Manley, P. W., Jahnke, W., and Grzesiek, S. (2008) Backbone NMR resonance assignment of the Abelson kinase domain in complex with imatinib. Biomol. NMR Assign. 2, 41–42. 49. Maniatis, T. (1982) Molecular cloning : a laboratory manual / T. Maniatis, E.F. Fritsch, J. Sambrook, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 50. Marley, J., Lu, M., and Bracken, C. (2001) A method for efficient isotopic labeling of recombinant proteins. J. Biomol. NMR 20, 71–75.

Chapter 4 Isotope Labeling in Mammalian Cells Arpana Dutta, Krishna Saxena, Harald Schwalbe, and Judith Klein-Seetharaman Abstract Isotope labeling of proteins represents an important and often required tool for the application of nuclear magnetic resonance (NMR) spectroscopy to investigate the structure and dynamics of proteins. Mammalian expression systems have conventionally been considered to be too weak and inefficient for protein expression. However, recent advances have significantly improved the expression levels of these systems. Here, we provide an overview of some of the recent developments in expression strategies for mammalian expression systems in view of NMR investigations. Key words: Isotope labeling, Nuclear magnetic resonance, Recombinant protein expression, Human embryonic kidney cells

1. Introduction: Mammalian Cell Expression System 1.1. Principle of Mammalian Cell-Mediated Protein Expression

Mammalian cell expression systems are being increasingly used to express proteins for structural biology. This is evident from the increase in the number of structures available in the Protein Data Bank (PDB) (1) of proteins purified from such sources. The main reason for switching to these higher eukaryotic expression systems is the ability to produce biologically active cell surface receptors and secreted glycoproteins. The functionality of these proteins is linked to the requirement for posttranslational modifications, such as glycosylation and disulfide bond formation, which can often only be satisfied by using mammalian systems and not other eukaryotic systems, such as yeast and insect cells. Protein expression in mammalian cells involves transfection with a plasmid carrying the gene of interest under the control of a mammalian promoter. The mammalian gene can be introduced

Alexander Shekhtman and David S. Burz (eds.), Protein NMR Techniques, Methods in Molecular Biology, vol. 831, DOI 10.1007/978-1-61779-480-3_4, © Springer Science+Business Media, LLC 2012

55

56

A. Dutta et al.

into cells either by way of transient transfection or by developing a stable cell line. The former can be achieved with both adherent and suspension cells using polyethyleneimine (PEI). The only advantage of this method over stable cell line preparation is that milligram quantities of protein can be prepared in a few days time compared to the months required by the latter method (2–4). However, there are several disadvantages associated with the transient transfection approach (1) Not every protein, in particular membrane proteins, give high yields when transiently transfected; (2) Very large quantities of DNA need to be prepared; (3) The process needs to be repeated every time protein is needed, adding labor and cost. Stable cell line creation overcomes these limitations, although 2–6 months are required to establish a high-level expression system. The effort is often justified, however, by the advantages of a stable mammalian cell line over a transient transfect (1) The yield is typically very high; (2) The process becomes fast and robust once the cell line is created for a particular protein; (3) Stable cell lines are usually created using calcium phosphate transfection, an efficient and inexpensive method; (4) Cell lines can be established in cellular backgrounds that are specifically tailored to the needs of the protein being expressed. For example, heterogenous glycosolyation is a problem in NMR studies which typically require highly homogenous material. To overcome this problem, cell lines can be used that are deficient in complex heterogenous glycosylation capabilities (5); (5) The ability to create inducible cell lines is an additional advantage. In cases where constitutive expression of a particular protein is toxic to cells, placing the gene under an inducible promoter can allow expression of otherwise toxic proteins; (6) Finally, stable cells can be easily scaled up by transferring them to suspension cultures in spinner flasks or bioreactors. Episomal vectors developed from viruses, such as Epstein–Barr virus (EBV) (6), bovine papilloma virus (BPV) (7), BK virus (BKV) (8), and Simian virus 40 (SV40) (9) are mainly used for transfection. A list of commonly used vectors can be found in (10). The advantages of using episomal vectors over integrating genes into the DNA of the host cell are numerous (1) Episomal expression will result in gene expression independent of the regulatory mechanisms of the host cell and the position of integration in the host genome, leading to higher expression levels since these factors may unfavorably influence expression. Random integration may also disrupt the characteristics of the gene of interest (11); (2) There is no interruption in host cell gene expression, which is often the case with integrative vectors, the latter leading to transformation of the host cell and undesirable effects on protein production; (3) Episomal vectors can exist in multiple copies in a cell, thus leading to amplification of the gene of interest. The main criteria for selecting a suitable episomal vector are (1) high copy number in E. coli for large-scale DNA production; (2) a strong mammalian promoter

4

Isotope Labeling in Mammalian Cells

57

for high expression levels of the gene of interest in mammalian cells; and (3) small size so that genes of different lengths can be easily cloned into the vector. Thus, the vector typically consists of a strong mammalian promoter to drive expression of the mammalian gene of interest, a viral origin of replication activated by viral early genes, which is required to carry out its replication thereby maintaining the vector in the host system, and a eukaryotic selection marker, usually an antibiotic resistance gene, such as neomycin, to select for transfected cells. The human cytomegalovirus promoter has been shown to be very powerful in both HEK293 and Chinese Hamster Ovary (CHO) cells and is also active in most mammalian cells (12). 1.2. Mammalian Cell Lines

Mammalian cell lines used for transfection with virus-based vectors carrying the gene of interest usually already carry vectors encoding viral early genes corresponding to the origin of replication used by the new vector. Some of these early genes are SV40 T antigen, EBNA-1, E1, and E2 which bind to SV40, EBV, and BPV ori, respectively. The early genes act in trans to initiate replication by binding to the ori and they also act as enhancers, thereby increasing the copy number of viral vector and the expression levels of the protein of interest (13). Since very high copy numbers of the viral genes can lead to host cell death, regulation of viral replication can be imposed by transfecting mammalian cells with a different vector carrying the early genes (13). Mammalian cell lines that are widely used for protein expression are CHO and Human Embryonic Kidney 293 (HEK293) cells. Both are suitable for use in adherent and suspension cultures. The advantage of using CHO cells is the availability of various auxotrophs that can be used as selection markers for transfection. An example of such an auxotroph is dihydrofolate reductase (DHFR) deficient cells which are triple auxotrophs for hypoxanthine, glycine, and thymidine (14). Transfection of foreign genes along with DHFR genes in these cells allows for the selection of clones in a medium devoid of the above nutrients. Another advantage of this system is that it helps amplify the foreign gene when DHFR deficient cells are grown in the presence of methotrexate, which blocks DHFR activity. This causes the transfected cells to deal with low DHFR activity by amplifying the copy number of DHFR, thus amplifying the copy number of the transfected gene of interest. The disadvantages of the CHO cell line is that these cells do not carry all of the sugar transferring enzymes (15) that are present in human cell lines. This may lead to the production of functionally nonrelevant proteins. Further, some of the posttranslational modifications of human proteins are not appropriately carried out in these cells (16). It has also been seen that expression levels are usually lower in CHO cells than in HEK293 cells (12, 17). Therefore, the human cell line, HEK293, is used for proteins that require functionally sensitive posttranslational modifications.

58

A. Dutta et al.

There are two mutant cell lines of HEK293, HEK293E, expressing EBNA-1, and HEK293T, expressing SV40 large T antigen, that increase the copy number of plasmids with EBV and SV40 origins of replication, respectively, thereby increasing expression levels of the protein of interest (13). 1.3. Expression and Labeling of Recombinant Proteins in Mammalian Cells

Uniform isotope labeling of proteins expressed in mammalian cells is still under development (18). This is because mammalian cells, like insect cells, are unable to grow in the type of minimal media that are used for bacteria where glucose or glycerol and ammonium chloride are the only sources of carbon and nitrogen, respectively. Higher eukaryotes, like the organisms they represent, require certain essential amino acids, without which they are unable to grow. Thus, the use of simple sources for 13C and 15N isotope labeling of proteins in bacteria is not possible for mammalian cells. A number of recent approaches, however, have been developed to address this complication. The first uniformly isotope-labeled protein purified from mammalian cells was obtained by Hansen et al. (19) followed by (20, 21). The method involved purification of a mixture of isotope labeled amino acids from an acid hydrolysate of algae or bacteria grown in 15NH4Cl and 13C glucose or 13CO2 to prepare uniformly labeled urokinase in a Sp2/0 mouse myeloma cell line. Different purification protocols of the labeled mixture were tested but only acid hydrolysis, which removes bacterial and algal products that are toxic to mammalian cells, proved to work. The mixture, however, needed to be supplemented with commercially available amino acids that would degrade during hydrolysis, particularly glutamine and cysteine. A commercially available form of 15 N-cysteine was used. Because of the high cost involved in purchasing 15 N-glutamate, 15N-, and 15N/13C-glutamine were synthesized from labeled glutamate (available commercially), 15NH4Cl, and ATP. Dialyzed serum was used to prevent dilution of isotope labeled amino acids with unlabeled amino acids from the serum. Such purified amino acid mixtures are commercially available today. One such commercially available mixture from Martek Biosciences Corp. was tested to support expression in CHO cells (20). The growth medium was optimized in terms of its concentration, removal of amino acids, such as aspartic acid and asparagine, and addition of amino acids, such as arginine, cysteine, and glutamic acid after isolating and purifying from the mixture so that the highest level of protein expression was obtained. However, there are serious concerns regarding the toxic effects of feeding these mixtures to mammalian cells, leading to extensive methods of purification and optimization, and making it a time and cost inefficient method. HEK293 cells, the most commonly used system of expression for preparing isotope-labeled samples, have, to date, not been grown successfully in the presence of these mixtures (Klein-Seetharaman, unpublished results). An uniformly labeled 15N TGFb1 sample was prepared from CHO cells by

4

Isotope Labeling in Mammalian Cells

59

growing them in Minimum Essential Medium (MEM, Gibco) with dialyzed serum, uniformly 15N labeled choline and uniformly 15 N-labeled amino acids (except tryptophan which was labeled only in the backbone nitrogen) (22), demonstrating the utility of CHO systems. The total cost involved was $1,000/L. Therefore, this method could prove to be expensive if the protein to be labeled does not have high expression levels, as is the case with most membrane proteins. Mammalian growth media containing a mixture of certain isotope-labeled amino acids are commercially available and have been recently used for labeling the G-protein coupled receptor, bovine rhodopsin, using 15N labeled medium in which GKLQSTVW amino acids were 15N labeled (23). If all the amino acids in the mixture do not cover the complete sequence of the protein to be labeled, then this method may not lead to complete labeling, as was the case with rhodopsin where 50% of amino acids were labeled (23). Apart from the high costs involved, an additional problem is the inability to perform perdeuteration because of the sensitivity of mammalian cells to deuterium oxide. Therefore, the most widely used method to obtain structural information on mammalian proteins by NMR has been by conducting amino acid type selective (AATS) isotope labeling. AATS labels a protein with specific isotope-labeled amino acids rather than uniform labels and yields structural information selectively for the amino acid(s) used in labeling. This has been successfully done with numerous proteins (24–26). Extensive AATS labeling has been carried out with rhodopsin, which was labeled by using 15N-isotopes of tryptophan (26), lysine (25), histidine (27) and 13C-isotopes of tryptophan (27), histidine (27) and glutamate (28), all through expression in HEK293 cells. 15N-lysine, 13C-glycine/serine double labeling has also been shown for rhodopsin (25). For example, multiple combinations of 15N- and 13C-isotope-labeled rhodopsin samples were used to assign 15N-tryptophan resonances in the NMR spectrum of rhodopsin, shown in Fig. 1 (29). Since the protocol for such a labeling method has been optimized and works very well at least for rhodopsin, we are providing a step-by-step protocol below. The first step is the creation of an inducible stable cell line of rhodopsin in HEK293 cells. Scaling up the expression level is accomplished by growing these cells in a suspension culture, thereby producing the milligram quantities required for NMR studies. The yield of rhodopsin from such a transient transfection is on the order of 50 mg/15 cm2 plate. After establishing the stable cell line with the opsin (apo form of rhodopsin) gene, the next step is to transfer these cells to suspension culture and grow them in isotope-labeled medium containing unlabeled amino acids along with the labeled amino acid(s) of interest. Up to 10 mg of rhodopsin can be obtained from such suspension cultures when amino acid supplements are provided (30), but typically 2 mg of rhodopsin are obtained from

60

A. Dutta et al.

Fig. 1. Dephased (red line) and nondephased (blue line) 15N detected 13C/15N CP REDOR spectra of selectively labeled rhodopsin in DOPC lipid bilayers at 220 K. (a) a,e-15N-Trp; (b) 13C¢-Leu/a,e-15N-Trp; (c) 13U-Thr/a,e-15N-Trp; (d) 13C¢-Cys/a,e15 N-Trp; (e) 13C¢-Pro/a,e-15N-Trp; (f) 13C¢-Gly/a,e-15N-Trp. Figure reproduced with permission from (29).

4

Isotope Labeling in Mammalian Cells

61

isotope labeled medium in which amino acid mixtures cannot be supplemented, to avoid dilution of the isotope-labeled amino acids provided in the medium.

2. Materials 2.1. Expression of Isotope-Labeled Recombinant Proteins

1. Plasmid containing the gene of interest (see Note 1). 2. HEK293 cells. 3. Dulbecco’s modified Eagle medium (DMEM F-12). 4. Blasticidin (500 mg/mL stock solution prepared in DMEM F-12). 5. Geneticin, G418 (100 mg/mL stock solution prepared in DMEM F-12). 6. 0.05% Trypsin–EDTA (Gibco). 7. Penicillin–streptomycin (PS): 100 U/mL of each. 8. Fetal bovine serum (FBS). 9. Phosphate buffer saline (PBS): Autoclaved. 10. Tetracycline: 200 mg/mL (100× stock). 11. Sodium butyrate: 500 mM (100× stock), filtered. 12. Complete medium: DMEM F-12, 10% FBS, 1% PS. 13. Induction medium: Complete medium containing 2 mg/mL tetracycline and 5 mM sodium butyrate. 14. Selection medium: Complete medium containing 5 mg/mL blasticidin and 1, 2, or 3 mg/mL of G418. 15. Cryo medium: Complete medium containing 10% DMSO. 16. 2.5 M CaCl2. 17. BES: 50 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, pH 7.2, 250 mM NaCl, 1.5 mM Na2HPO4, 1 M NaOH used to adjust pH. 18. 15- and 10-cm2 Tissue culture plates. 19. 6- and 24-Well cell culture plates. 20. Sterile forceps. 21. Cloning rings. 22. Vacuum grease. 23. Dodecyl maltoside.

2.2. Specific Isotope Labeling of Proteins

Suspension DMEM medium: 1. Table 1 gives the individual components of suspension DMEM in their final concentrations based on the composition of commercially available DMEM. 100× stock solutions are prepared

mg/L

84

42

105

105

146

30

66

95

16

94

Essential amino acid

Arginine  HCl

Histidine  HCl  H2O

Isoleucine

Leucine

Lysine  HCl

Methionine

Phenylalanine

Threonine

Tryptophan

Valine

42 104

Tyrosine  2Na+  2H2O

None

30

584

Serine

Proline

Glycine

Glutamine

None

63

Cystine  2HCl Glutamate

None

None

None

mg/L

Aspartate

Asparagine

Alanine

Nonessential amino acid

Table 1 Amino Acid Composition of suspension DMEM

pantothenate

0.4 4

Thiamine  HCl

4

Pyridoxal  HCl Riboflavin

4

7.2

4

4

4

mg/L

Niacinamide

i-Inositol

Folic Acid

Choline Chloride

D-Ca

Vitamins

Glucose

Phenol Red  Na+

NaH2PO4  H2O

NaCl

KCl

MgSO4

Fe(NO3)3  9H2O

CaCl2

Inorganic salts and other

4,500

15

125

6,400

400

97.7

0.1

50

mg/L

62 A. Dutta et al.

4

Isotope Labeling in Mammalian Cells

63

for most components (see Note 2). Each component is dissolved in distilled water. 2. 0.05% trypsin–EDTA (Subheading 2.1). 3. FBS (Subheading 2.1). 4. PS (Subheading 2.1). 5. Dialyzed FBS: Heat inactivated, dialyzed against PBS and then filtered. 6. 10% Pluronic F-68 (100× stock), filtered. 7. 5 mg/mL Heparin (100× stock), filtered. 8. 15-cm2 tissue culture plates. 9. 2-L spinner flasks. 10. 100× tetracycline (Subheading 2.1). 11. 100× sodium butyrate (Subheading 2.1). 12. 20% (w/v) glucose, sterile filtered. 13. 8% (w/v) NaHCO3. 14. Complete medium (Subheading 2.1). 15. Selection medium (Subheading 2.1).

3. Methods 3.1. Expression of Isotope-Labeled Recombinant Proteins

Following is a protocol for generating an inducible stable cell line of HEK293 (30–32). 1. Wake up HEK293 cells from cryostocks and maintain them in 15 cm2 plates in 25 mL of complete medium containing 5 mg/mL blasticidin. 2. Split the cells into a 10-cm2 dish at 80% confluency. 3. Split the cells 1:10 or 1:8 (~1–2 million cells per plate) into complete medium containing 5 mg/mL blasticidin the day before transfection. 4. On the day of transfection, the cells should be 30–40% confluent. To transfect the cells, prepare the following cocktail, adding components in the order given (volumes are for one 10-cm2 plate): In a falcon tube, mix 30 mg of plasmid DNA, 50 mL of 2.5 M CaCl2, 500 mL of BES, and sterile water to 1 mL. Incubate the mixture for exactly 1 min and gently add it to the cells. After 1 h, verify the efficiency of transfection by the presence of calcium phosphate precipitate which appears as fine sand particles between cells. 5. Incubate the plates at 35°C and 3% CO2 for 19 h.

64

A. Dutta et al.

6. After 19 h, wash the plates twice with complete medium containing 5 mg/mL blasticidin and add fresh complete medium containing 5 mg/mL blasticidin. 7. Incubate the cells at 37°C and 5% CO2 overnight. 8. Split the cells 1:10 (0.5–1 million cells/plate) into nine plates, three plates each for selection with G418 (1, 2, and 3 mg/mL). 9. Replace the medium after 20 h with selection medium. 10. Replace the selection medium every 2–3 days until colonies of workable size are formed. 11. On the day of picking the clones: (a) Circle the colonies that are to be picked such that the circles are big enough to place the cloning rings on its perimeter. (b) Aspirate the medium and look for more colonies on the empty plate by eye. Wash with 8 mL of PBS. (c) Using sterile forceps, dip one end (base) of each cloning ring in vacuum grease and place it over each clone (see Note 3). Make sure there is no grease on the inner walls of the rings. Make sure that the rings form a well around the clones with a leak proof seal at the bottom. (d) Add 40 mL of 0.05% trypsin–EDTA to each well and incubate for 1 min. Then, add 80 mL of fresh selection medium and gently pipette up and down. Transfer the cells (100 mL) to a 24-well plate containing 1 mL of selection medium per well. (e) Repeat for all the clones. 12. Replace the selection medium every 2–3 days until the cells are confluent. Make a note of medium changes and transfers to 6-well plates in a chart (see step 13 for details). 13. As the cells become confluent, transfer them to two 6-well plates at different cell densities. For the transfer: (a) Pipette 3 mL of selection medium into each well of a 6-well plate. (b) Aspirate the medium from the cells (24-well plate) and add 300 mL of 0.05% trypsin–EDTA to each well and incubate for 1 min. (c) Add 1,200 mL (use a 1-mL pipette and add 600 + 600 mL) of selection medium and gently pipette up down. (d) Remove 300 mL and add it to one well and place the remaining (1,200 mL) in another well. The well with higher cell density will be induced in the future to screen the clones while the one with lower density will be maintained and used for making glycerol stocks.

4

Isotope Labeling in Mammalian Cells

65

14. Change the selection medium on the cells every 2–3 days. 15. As soon as the cells in the higher density well reach confluence, induce with induction medium. 16. Harvest the cells 48-h post induction. (a) After harvesting, solubilize the cells in a detergent suitable for the membrane protein of interest (e.g., 1% dodecyl maltoside). (b) Centrifuge the solubilized cells at 126,000 × g for 20 min at room temperature and collect the supernatant. 17. Check for levels of expression by performing a Western dot blot on serial dilutions of the supernatant in PBS, containing 1% of the detergent used for solubilization, versus protein samples of known concentrations (see Note 4). 18. When the cells from the low density well reach confluence, split 1:5 into two 10-cm2 cell culture dishes. Add 0.5 mL of 0.05% trypsin–EDTA to a 6-well plate, and then add 1.5 mL of selection medium. Add 1 mL of this cell suspension to each of the two 10-cm2 plates. 19. After the cells reach confluence on the 10-cm2 plate, prepare three 1-mL glycerol stocks from each plate. To prepare glycerol stocks: (a) Wash 70–90% confluent plates twice with PBS and trypsinize with 1 mL of 0.05% trypsin–EDTA for 1 min. (b) Add 10 mL of complete medium (no selection) and collect the cells in a 15-mL falcon tube. (c) Centrifuge the cells at 600 × g for 10 min at 4°C. (d) Aspirate the medium and gently resuspend the cells in 3 mL of cryo medium. (e) Transfer the cells to cryo vials in 1-mL aliquots and place the tubes in cryo boxes. Incubate at −20°C for 1 h, and then −80°C overnight. (f) On the following day, move the tubes to liquid nitrogen storage tanks. 3.2. Specific Isotope Labeling of Proteins

After estimating the yield from above (Subheading 3.1) and identifying the highest yield clone, the following procedure is used to prepare a protein sample, in suspension culture, in which particular amino acids are specifically isotope labeled (30–32). 1. Grow the highest yielding stable cell line clone in complete medium. 2. Split the cells 1:5 into 10-cm2 dishes after ~3 days using 15 mL of selection medium.

66

A. Dutta et al.

3. Split the cells until a sufficient number of plates are obtained for setting up spinner flasks. For reference, 1 mg of protein per liter of suspension culture is usually obtained for rhodopsin and 3–4 confluent 15-cm2 plates are used to inoculate a 500-mL flask (see Notes 5 and 6). 4. Prepare labeled suspension DMEM medium from 100× stocks of the individual components (Table 1, Subheading 2.2), except for the isotope labeled amino acid(s). Supplement with 10% dialyzed FBS and 1% PS or obtain isotope-labeled medium from a commercial source. (a) Add glucose, NaCl, glutamine, isotope-labeled amino acid(s) as solids. (b) Lower the glutamine concentration to half when starting the suspension culture. (c) Add Pluronic F-68 and heparin to 1× concentration. (d) Add isotope-labeled amino acid(s) as solids. 5. Inoculate the suspension cultures and grow at 37°C, spinning at 47 rpm for 6 days. To inoculate, add 2 mL of 0.05% trypsin– EDTA to each plate and extract the cells with 8 mL of unlabeled suspension medium. Collect cells from each plate (10-mL volume) and centrifuge at 1,000 × g for 10 min at 4°C. Aspirate the medium and add 10 mL of labeled suspension DMEM medium to each plate and resuspend the cells. Add the cell suspension to the suspension culture in the spinner flask. 6. After 6 days (see Note 7), supplement the growth medium with 6 mL of 20% (w/v) glucose and 4 mL of 8% (w/v) NaHCO3 and induce expression with 5 mM sodium butyrate and 2 mg/mL of tetracycline. 7. After the 6th day, feed the cells with 6 mL of 20% glucose every 24 h. 8. Grow the cells for 2 more days after the 6th day (total 8 days in suspension). 9. Harvest at the end of 8 days.

4. Conclusions A variety of expression systems have been developed that can be used to express a protein of choice in isotope-labeled form, each system having advantages and disadvantages. These systems include bacterial, yeast, insect, and mammalian cells. The choice of the system largely depends on the type of the protein to be expressed. Other factors are the costs involved, the need for modifications and

4

Isotope Labeling in Mammalian Cells

67

yield of protein required. Bacterial expression systems are most commonly used for soluble protein expression due to ease of use and cost-effectiveness in cloning and expression. However, this system is not the system of choice for proteins that require posttranslational modification for their activity. In such cases, eukaryotic expression systems are required. Yeast being both eukaryotic and a microorganism is a simple system to use, allowing relatively inexpensive complete isotope labeling of proteins for NMR studies. Although it can carry out some posttranslational modifications, these are often not sufficient and deficiency in folding machinery can be a problem for some proteins, especially membrane proteins. For these reasons, higher eukaryotic systems, such as insect cells have been in use where posttranslational modifications are very similar to those found in mammalian cells. The greatest advantage of insect cell expression is the very large amounts of proteins that can be expressed, in most cases more than that in mammalian cells. However, disadvantages of expensive isotope-labeled media, incomplete labeling and intolerability to perdeuteration that hamper mammalian cell expression are also prevalent in insect cells. Hence, efforts are ongoing towards further developing mammalian expression systems since these are the only systems in which the full complement of folding and posttranslational modification machineries are available, optimally supporting the activity of the expressed protein.

5. Notes 1. A tetracycline (tet)-regulated mammalian expression vector should be used. 2. All solutions were prepared as 100× concentrated stock solutions, except glucose, NaCl, glutamine, and the isotopelabeled amino acid(s), which were added as solids. Typically, 500 mL of stock solutions were prepared and filtered to maintain sterility. Sterile stock solutions were kept at 4°C and were stable for several weeks. 3. Cloning rings, vacuum grease, and forceps should be autoclaved. 4. PVDF membranes should not be used. 5. Cells should be counted before adding them to spinner flasks so that appropriate number of cells (in the range between 60 and 90 million cells/500 mL) can be transferred. 6. A 2-L spinner flask is ideal for a 500-mL suspension culture. 7. The color of the medium should turn yellow after 6 days.

68

A. Dutta et al.

References 1. Nettleship, J. E., Assenberg, R., Diprose, J. M., Rahman-Huq, N., and Owens, R. J. (2010) Recent advances in the production of proteins in insect and mammalian cells for structural biology. J. Struct. Biol. 172, 55–65. 2. Aricescu, A. R., Lu, W., and Jones, E. Y. (2006) A time- and cost-efficient system for highlevel protein production in mammalian cells. Acta. Crystallogr. D Biol. Crystallogr. 62, 1243–1250. 3. Lee, J. E., Fusco, M. L., and Ollmann Saphire, E. (2009) An efficient platform for screening expression and crystallization of glycoproteins produced in human cells. Nat. Protoc. 4, 592–604. 4. Nettleship, J. E., Rahman-Huq, N., and Owens, R. J. (2009) The production of glycoproteins by transient expression in mammalian cells. Methods Mol. Biol. 498, 245–263. 5. Reeves, P. J., Callewaert, N., Contreras, R., and Khorana, H. G. (2002) Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl. Acad. Sci. U.S.A. 99, 13419–13424. 6. Sclimenti, C. R., and Calos, M. P. (1998) EpsteinBarr virus vectors for gene expression and transfer. Curr. Opin. Biotechnol. 9, 476–479. 7. Sambrook, J., Rodgers, L., White, J., and Gething, M. J. (1985) Lines of BPV-transformed murine cells that constitutively express influenza virus hemagglutinin. EMBO J. 4, 91–103. 8. Grossi, M. P., Caputo, A., Rimessi, P., Chiccoli, L., Balboni, P. G., and Barbanti-Brodano, G. (1988) New BK virus episomal vector for complementary DNA expression in human cells. Arch. Virol. 102, 275–283. 9. Piechaczek, C., Fetzer, C., Baiker, A., Bode, J., and Lipps, H. J. (1999) A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells. Nucleic Acids Res. 27, 426–428. 10. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, CSH Laboratory Press, Cold Spring Harbor, NY. 11. Doerfler, W., Schubbert, R., Heller, H., Kammer, C., Hilger-Eversheim, K., Knoblauch, M., and Remus, R. (1997) Integration of foreign DNA and its consequences in mammalian systems. Trends Biotechnol. 15, 297–301. 12. Xia, W., Bringmann, P., McClary, J., Jones, P. P., Manzana, W., Zhu, Y., Wang, S., Liu, Y., Harvey, S., Madlansacay, M. R., McLean, K.,

Rosser, M. P., MacRobbie, J., Olsen, C. L., and Cobb, R. R. (2006) High levels of protein expression using different mammalian CMV promoters in several cell lines. Protein Expr. Purif. 45, 115–124. 13. Van Craenenbroeck, K., Vanhoenacker, P., and Haegeman, G. (2000) Episomal vectors for gene expression in mammalian cells. Eur. J. Biochem. 267, 5665–5678. 14. Urlaub, G., and Chasin, L. A. (1980) Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc. Natl. Acad. Sci. U.S.A. 77, 4216–4220. 15. Grabenhorst, E., Schlenke, P., Pohl, S., Nimtz, M., and Conradt, H. S. (1999) Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells. Glycoconj. J. 16, 81–97. 16. Suttie, J. W. (1986) Report of Workshop on expression of vitamin K-dependent proteins in bacterial and mammalian cells. Madison, Wisconsin, USA, April 1986, Thromb. Res. 44, 129–134. 17. Schlaeger, E. J., Kitas, E. A., and Dorn, A. (2003) SEAP expression in transiently transfected mammalian cells grown in serum-free suspension culture. Cytotechnology 42, 47–55. 18. Takahashi, H., and Shimada, I. (2010) Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells. J. Biomol. NMR 46, 3–10. 19. Hansen, A. P., Petros, A. M., Mazar, A. P., Pederson, T. M., Rueter, A., and Fesik, S. W. (1992) A practical method for uniform isotopic labeling of recombinant proteins in mammalian cells. Biochemistry 31, 12713–12718. 20. Lustbader, J. W., Birken, S., Pollak, S., Pound, A., Chait, B. T., Mirza, U. A., Ramnarain, S., Canfield, R. E., and Brown, J. M. (1996) Expression of human chorionic gonadotropin uniformly labeled with NMR isotopes in Chinese hamster ovary cells: an advance toward rapid determination of glycoprotein structures. J. Biomol. NMR 7, 295–304. 21. Shindo, K., Masuda, K., Takahashi, H., Arata, Y., and Shimada, I. (2000) Backbone 1H, 13C, and 15N resonance assignments of the anti-dansyl antibody Fv fragment. J. Biomol. NMR 17, 357–358. 22. Archer, S. J., Bax, A., Roberts, A. B., Sporn, M. B., Ogawa, Y., Piez, K. A., Weatherbee, J. A., Tsang, M. L., Lucas, R., Zheng, B. L., and et al. (1993) Transforming growth factor beta 1: NMR signal assignments of the recombinant protein expressed and isotopically enriched using Chinese hamster ovary cells. Biochemistry 32, 1152–1163.

4 23. Werner, K., Richter, C., Klein-Seetharaman, J., and Schwalbe, H. (2008) Isotope labeling of mammalian GPCRs in HEK293 cells and characterization of the C-terminus of bovine rhodopsin by high resolution liquid NMR spectroscopy. J. Biomol. NMR 40, 49–53. 24. Arata, Y., Kato, K., Takahashi, H., and Shimada, I. (1994) Nuclear magnetic resonance study of antibodies: a multinuclear approach. Methods Enzymol. 239, 440–464. 25. Klein-Seetharaman, J., Reeves, P. J., Loewen, M. C., Getmanova, E. V., Chung, J., Schwalbe, H., Wright, P. E., and Khorana, H. G. (2002) Solution NMR spectroscopy of (alpha -15N) lysine-labeled rhodopsin: The single peak observed in both conventional and TROSYtype HSQC spectra is ascribed to Lys-339 in the carboxyl-terminal peptide sequence. Proc. Natl. Acad. Sci. U.S.A. 99, 3452–3457. 26. Klein-Seetharaman, J., Yanamala, N. V., Javeed, F., Reeves, P. J., Getmanova, E. V., Loewen, M. C., Schwalbe, H., and Khorana, H. G. (2004) Differential dynamics in the G protein-coupled receptor rhodopsin revealed by solution NMR. Proc. Natl. Acad. Sci. U.S.A. 101, 3409–3413. 27. Patel, A. B., Crocker, E., Reeves, P. J., Getmanova, E. V., Eilers, M., Khorana, H. G., and Smith, S. O. (2005) Changes in interhelical hydrogen bonding upon rhodopsin activation. J. Mol. Biol. 347, 803–812.

Isotope Labeling in Mammalian Cells

69

28. Han, M., and Smith, S. O. (1995) Highresolution structural studies of the retinal– Glu113 interaction in rhodopsin. Biophys. Chem. 56, 23–29. 29. Werner, K., Lehner, I., Dhiman, H. K., Richter, C., Glaubitz, C., Schwalbe, H., KleinSeetharaman, J., and Khorana, H. G. (2007) Combined solid state and solution NMR studies of alpha,epsilon-15N labeled bovine rhodopsin. J. Biomol. NMR 37, 303–312. 30. Reeves, P. J., Kim, J. M., and Khorana, H. G. (2002) Structure and function in rhodopsin: a tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants. Proc. Natl. Acad. Sci. U.S.A. 99, 13413–13418. 31. Reeves, P. J., Callewaert, N., Contreras, R., and Khorana, H. G. (2002) Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line 452. Proc. Natl. Acad. Sci. U.S.A. 99, 13419–13424. 32. Reeves, P. J., Thurmond, R. L., and Khorana, H. G. (1996) Structure and function in rhodopsin: high level expression of a synthetic bovine opsin gene and its mutants in stable mammalian cell lines. Proc. Natl. Acad. Sci. U.S.A. 93, 11487–11492.

Chapter 5 Cell-Free Protein Production for NMR Studies Mitsuhiro Takeda and Masatsune Kainosho Abstract The cell-free expression system using an Escherichia coli extract is a practical method for producing isotope-labeled proteins. The advantage of the cell-free system over cellular expression is that any isotopelabeled amino acid can be incorporated into the target protein with minimal scrambling, thus providing opportunities for advanced isotope labeling of proteins. We have modified the standard protocol for E. coli cell-free expression to cope with two problems specific to NMR sample preparation. First, endogenous amino acids present in the E. coli S30 extract lead to dilution of the added isotope. To minimize the content of the remaining amino acids, a gel filtration step is included in the preparation of the E. coli extract. Second, proteins produced by the cell-free system are not necessarily homogeneous due to incomplete processing of the N-terminal formyl-methionine residue, which complicates NMR spectra. Therefore, the protein of interest is engineered to contain a cleavable N-terminal histidine-tag, which generates a homogeneous protein after the digestion of the tag. Here, we describe the protocol for modified E. coli cell-free expression. Key words: Cell-free synthesis, S30 extract from E. coli, NMR, Stable isotope labeling

1. Introduction The NMR study of a protein starts by expressing the isotope-labeled protein. It is now widely accepted that the cell-free expression system is one of the most practical methods available for producing isotope-labeled proteins, along with the cellular expression system (1, 2). The cell-free system employs a cell extract from an organism such as Escherichia coli (3–7), an extract from wheat germ (8) or a mixture of recombinant proteins (9). As a consequence of its reduced amino acid metabolic activity in vitro, isotope-labeled amino acids can be efficiently incorporated into a target protein with minimal scrambling (10), which generates new opportunities for advanced selective isotope labeling of proteins, such as stereoarray isotope-labeled proteins (11–13). Alexander Shekhtman and David S. Burz (eds.), Protein NMR Techniques, Methods in Molecular Biology, vol. 831, DOI 10.1007/978-1-61779-480-3_5, © Springer Science+Business Media, LLC 2012

71

72

M. Takeda and M. Kainosho

We utilize a cell-free system, in which the transcription and translation systems are reconstituted in a vessel by combining an E. coli extract and several components. The E. coli cell-free system has a long history and the level of expression has been improving due to extensive investigations. The E. coli cell-free protein expression system is generally regarded as a practical method for producing isotope-labeled proteins, along with the cellular expression system. E. coli extract is now commercially available. In many of the E. coli cell-free systems, transcription is mediated by T7 RNA polymerase, and thus any plasmid encoding a DNA sequence under the control of the T7 promoter, such as the pET vectors (Novagen), can be used. Therefore, it is now relatively easy to test for expression of proteins of interest by using the E. coli cell-free system. We encountered two problems in the standard protocol of the E. coli cell-free procedure in terms of NMR sample preparation, and thus addressed the problems (14). First, the endogenous amino acids present in the E. coli S30 extract are incorporated into the target protein, which leads to isotope dilution. When (2H, 13C, 15 N)-calmodulin (CaM) protein was produced by using an E. coli S30 extract prepared by the standard protocol, the labeling efficiency was only ~90%. Therefore, to minimize the residual amino acids, we introduced a gel filtration step into the protocol for preparing the E. coli S30 extract. As a result, the labeling efficiency increased to 96% without loss of activity in the extract (14). Second, the formyl group of the N-terminal methionine in proteins produced by the cell-free system is not processed completely. The presence of the formyl moiety causes large, chemical shift changes in nearby residues, giving rise to doubled peaks, which correspond to formylated and deformylated forms. For example, the NMR signals from CaM, which has a flexible N terminus, are affected by the heterogeneity over an extensive region (14). Larger spectral complications may arise from heterogeneity of the peptide chain in proteins with a structured N terminus. We solved this problem by engineering the target protein with a cleavable N-terminal tag. After cleaving the tag, the protein was homogeneous, and the (1H, 15N) heteronuclear single quantum correlation (HSQC) spectrum of this CaM preparation lacked detectable extra peaks (14). To engineer proteins with an N-terminal cleavable tag, a template vector encoding an N-terminal tag followed by the multicloning site is useful. We constructed a family of vectors by modifying a pIVEX2.3d vector (Roche) to encode the N-terminal tag. In these vectors, the amino acid sequences are identical, but the DNA sequence in the region of the N-terminal tag is different. Introducing silent mutations into the DNA sequence in the N-terminal region can lead to different expression levels of the target protein (14). Therefore, plasmids with a higher expression level can be isolated when the target DNA sequence is introduced into the modified

5

Cell-Free Protein Production for NMR Studies

73

pIVEX vectors. In the case of CaM, for example, improvements of the protein yields relative to original sequence were observed for two of the ten pIVEX vectors. In the large-scale reaction performed with the best sequence and 51.1 mg of amino acid mixture (1.7 mg/mL), the amount of CaM synthesized was 5.2 mg (10 wt%). This was twice the yield from the construct prior to the silent mutagenesis (14).

2. Materials 2.1. Preparation of E. coli S30 Extract

1. RNase-free Milli-Q PF Plus water (Millipore) (see Note 1). 2. LB medium: Dissolve 20 g of Luria Broth powder in 1 L of water. 3. Incomplete rich medium: Dissolve 5.6 g of KH2PO4, 28.9 g of K2HPO4, 1 g of Bacto yeast extract, and 1.5 mg of thiamine hydrochloride in 1 L of water, and autoclave the solution. Cool to room temperature and add 50 mL of 40% (w/v) D-glucose and 10 mL of 100 mM Mg(OAc)2. 4. BL21 Star (DE3) cells (Invitrogen) (see Note 2). 5. French press (see Note 3). 6. S30 buffer: Combine 10 mL of 1 M Tris–OAc, pH 8.2, 10 mL of 1.4 M Mg(OAc) 2, and 10 mL of 6 M KOAc in a final volume of 1 L of RNase-free water, atuoclave, and add 1 mL of 1 M DTT. 7. Gel filtration apparatus: Pack Sephadex G25 resin into a 2.5 × 20-cm chromatography column and set the column vertically at 4°C. After the resin settles, attach a funnel to the top of the column and equilibrate with 500 mL of S30 buffer (see Note 4). 8. Dialysis tubing (molecular weight cutoff: 6–8 kDa). 9. Polyethylene glycol (PEG)-8000: Avg. MW 8,000 Da. 10. 2-mercaptoethanol. 11. Diethyl pyrocarbonate (DEPC). 12. Centrifugation tube: Wash the tubes with DEPC water and autoclave to completely remove RNase. 13. 40% (w/v) D-glucose. 14. 100 mM Mg(OAc)2. 15. 1 M Tris–OAc, pH 8.2. 16. 1.4 M Mg(OAc)2. 17. 6 M KOAc. 18. 1 M DTT.

74

M. Takeda and M. Kainosho

2.2. Preparation of T7 RNA Polymerase

1. M9 medium supplemented with tryptone peptone: Combine 1 g of Tryptone peptone (Bectone), 5 g of NaCl, 1 g of NH4Cl, 3 g of KH2PO4, and 6 g of Na2HPO4, and add water to 1 L. After autoclaving, add 10 mL of sterile-filtered 40% (w/v) glucose and 1 mL of sterile 1 M MgSO4. 2. Sonication buffer A: 50 mM Tris–HCl, pH 8.1, 20 mM NaCl, 2 mM EDTA, and 1 mM DTT. 3. Sonication buffer B: 50 mM Tris–HCl, pH 8.1, 20 mM NaCl, 2 mM EDTA, 1 mM DTT, 1.5 mg/mL lysozyme, 20 μg/mL PMSF, 10 μg/mL bacitracin, and 0.1 mM benzamidine. 4. Dialysis buffer: 20 mM sodium phosphate–NaOH, pH 7.7, 1 mM EDTA, 1 mM DTT, and 5% glycerol. 5. SP-Sepharose Fast Flow (GE Healthcare): This resin is packed into a column (3 × 30 cm). 6. Elution buffer: 20 mM sodium phosphate–NaOH, pH 7.7, 1 mM EDTA, 1 mM DTT, 5% glycerol, 200 mM NaCl, and 20 μg/mL PMSF. 7. Isopropyl-beta-D-thiogalactopyranoside (IPTG). 8. 0.8% (w/v) sodium deoxycholate. 9. 2 M Ammonium sulfate. 10. 10% (w/v) Polyethleneimine–HCl, pH 7.0. 11. Saturated ammonium sulfate: Adjust pH to 7.0 using 1 M Tris. 12. E. coli strain BL21 (DE3) transformed with a plasmid encoding T7 RNA polymerase. 13. LB medium (Subheading 2.1). 14. 40% D-Glucose (Subheading 2.1). 15. 1 M MgSO4: Sterilize by autoclaving.

2.3. Cell-Free Synthesis Reaction

1. LM mixture: To prepare the low-molecular-weight mixture, combine 22 mL of 2 M HEPES–KOH, pH 7.5, 33.4 mL of 6 M K(OAc), 210 mg of DTT, 530 mg of ATP, 338 mg of CTP, 335 mg of GTP, 310 mg of UTP, 172 mg of cAMP, 28 mg of folinic acid, and 140 mg of tRNA, and, if needed, 64 mL 50% (w/v) PEG-8000 (see Note 5), and then add RNase-free water up to 200 mL (see Note 6). 2. Dialysis membrane for cell-free reaction: A size 8 cellulose membrane (Viskase Sales Corporation) ensures a reproducible expression level. Cut the membrane to an appropriate size. First, soak the membrane in RNase-free water and then warm it to 70–80°C. After it has cooled down, wash the membrane with RNase-free water. Second, soak the membrane in water containing ~50 mM of NaHCO3, warm it in a microwave to remove glycerol, and then wash it with RNase-free water. Third, soak the membrane in water containing ~1 mM EDTA, warm it in a microwave to further remove glycerol, and then

5

Cell-Free Protein Production for NMR Studies

75

wash it with RNase-free water three times. Finally, soak the membrane in water and autoclave it. 3. 0.645 M Creatine phosphate (see Note 7). 4. 10 mg/mL Creatine kinase (see Note 7). 5. RNase inhibitor (Porcine liver). 6. 1.4 M NH4OAc or (15N) NH4OAc (see Note 8). 7. T7 RNA polymerase at ~11 mg/mL (see Note 9). 8. Template DNA: A plasmid containing the sequence encoding the target protein at ~1 mg/mL (see Note 10). 9. 2× Loading buffer: 2 mL of 0.5 M Tris–HCl, pH 6.8, 4 mL of 10% sodium dodecyl sulfate (SDS), 1.2 mL of 2-mercaptoethanol, 2 mL of glycerol, 1–2 mg of bromophenol blue, and 0.8 mL of water. 10. RNase-free water (Subheading 2.1). 11. 0.5 M Mg(OAc)2. 12. S30 extract (Subheading 3.1). 13. Reaction solution: Mix 112 μL of RNase-free water, 9.8 μL of 1.4 M NH4OAc, 15 μL of 0.5 M Mg(OAc)2, 20 μL of SAIL amino acids, 40 μL of 0.645 M creatine phosphate, 125 μL of LM mixture, 10 μL of 1 mg/mL template DNA, 4.5 μL of 11 mg/mL T7 RNA polymerase, 1.25 μL of 40 U/mL RNase inhibitor, 12.5 μL of 10 mg/mL creatine kinase, and 150 μL of S30 extract for 0.5 mL total volume. 14. Dialysis solution: Mix 1160.8 μL of RNase-free water, 39.2 μL of 1.4 M NH4OAc, 60 μL of 0.5 M Mg(OAc)2, 80 μL of SAIL amino acids, 160 μL of 0.645 M creatine phosphate, and 500 μL of LM mixture for 2 mL total volume. 15. Phosphate buffer: 10 mM potassium phosphate, pH 7.0. Adjust pH using NaOH or HCl. 16. 6 M KOAc (Subheading 2.1). 17. 50% (w/v) PEG-8000. 18. 10% SDS. 19. 0.5 M Tris–HCl, pH 6.8. 20. 2-Mercaptoethanol.

3. Methods The E. coli S30 extract preparation protocol essentially follows previously reported procedures (3, 7, 15), but contains a gel filtration step to minimize the endogenous amino acids. Typically, preparation of the S30 extract takes 2–3 days for one person. We strongly recommend saving an aliquot of the extract at each step to monitor

76

M. Takeda and M. Kainosho

the activity throughout the purification. T7 RNA polymerase is also prepared following established methods (16–18). The preparation of T7 RNA polymerase takes 1 week for one person. The cell-free expression strategy for new proteins is shown in Fig. 1 . As the first step, we recommend starting with E. coli cellular expression of the target protein with uniform 15N labeling.

Fig. 1. Flowchart of the cell-free strategy (reproduced from ref. 14 with permission from Elsevier Science).

5

Cell-Free Protein Production for NMR Studies

77

It is less costly and more efficient to produce proteins from E. coli cells than by using the cell-free method, and cellular expression is better suited for optimizing the purification protocol and the NMR buffer conditions. In general, a protein that exhibits poor cellular expression is also likely to show poor cell-free expression, unless the protein produced is toxic to E. coli cells. Next, several parameters are optimized to maximize the expression level in the cell-free reaction based on small-scale reactions under different conditions. There are various parameters to consider in the optimization process, including the concentration of magnesium, incubation temperature, incubation time, and concentration of PEG. If necessary, the target DNA sequence can be transferred to the line of pIVEX vectors with different silent mutations, as described in the introduction. When an acceptable expression level is attained, a large quantity of the 15N-labeled protein is produced and its (1H, 15N)-HSQC spectrum is compared with that of the protein produced by cellular expression. This comparison helps to detect any possible difference between in vivo and in vitro expression. Special attention should be paid to the incomplete deformylation of the N terminus of proteins in the cell-free reaction. The presence of formylated and deformylated species complicates NMR spectra. To overcome this problem, the use of an engineered N-terminal cleavable tag is helpful, as shown in Fig. 2. 3.1. Preparation of E. coli S30 Extract

1. Inoculate 10 mL of LB medium with the E. coli stock cells (BL21 star (DE3) strain) and incubate the preculture overnight at 37°C with shaking. 2. Add the preculture to 1 L of incomplete rich medium prewarmed to 37°C, and incubate the culture at the same temperature with shaking to an optical density at 600 nm (OD600) of 0.7 (see Note 11). 3. Centrifuge the cells at 5,000 × g for 10 min at 4°C and then wash them three times with 200 mL of ice-cold S30 buffer supplemented with 0.05% 2-mercaptoethanol (see Note 12). Store the cells as a pellet at −80°C. 4. Gently resuspend the cell pellet in 200 mL of ice-cold S30 buffer supplemented with 0.05% 2-mercaptoethanol. Centrifuge at 5,000 × g for 10 min at 4°C and weigh the pellet. Resuspend the pellet in 1.27 mL of S30 buffer per gram of cells. 5. Disrupt the cells with a French Press at 20,000 psi (1,400 kg cm−2) (see Note 13). Immediately after the disruption, add 30 μL of 1 M DTT and centrifuge the lysate at 30,000 × g for 30 min at 4°C in DEPC-treated/autoclaved centrifuge tubes. Carefully collect ~1.4 mL of supernatant per gram of E. coli without mixing with the precipitate.

M. Takeda and M. Kainosho

a

105

110

Q3E2

Q3E2

T5 Q3E2

Q3E2

T70 D2 D2

115

T70 R74

R74 I9 I9

Q3 Q3 K77 K77

D78 D78

M71 M71

A73 A73

K13 K13

120

15N (p.p.m.)

T5

125 A1

L4

L4

M0

130

11

10

9

8

6

7

1H (p.p.m.)

b

105 G113 G33 G59

G40

N137D2

N137D2

Q135E2

Q135E2

T62 G96

N53D2

T5 G98 G25

G134

T29 T44

G61

G132 V55 G23 Q8E2 Q41E2 T146

D95

T117

T26 D58

T110

N53D2

N111D2

Q143E2 N42D2 S17

Q3E2 Q49E2

110

Q41E2 Q8E2 N111D2 Q143E2 N42D2 Q3E2 Q49E2

D22 M145 T79 N60D2

T70 F19 Q135 N60D2 E127 N97D2 R90 E54 M109 Y99 N97D2 M72 D93 D131 D129 F92 S81 D20 R74 E139 D2E67 R106 Q49 N60 T34 N53 I125 M76 I52 Q143 Y138 A103 V91 Q3 M36 Q41 K75 F65 F89 L69 R126 E87 E47 H107 E119 A128 R37 E84 S38V108 L112 M51 F16 E11 D122 M124 V142 E140 E104 N97 E123 Q8 L32 E7 I9 E83D50 M144 L39 E6 E120 E45 F12 D24 L18 L48 E114 K77 E14 K30 D133 A46 A10 E31 D118 L105 V121 D56 R86 D78 M71 V35 A102 I85 A88 L4 F68 A73 E82 A15 N111 K13 D80 A1 K21 S101 I63 K115 K148 V136 F141 L116 K94 N42

D64 I100

115

T28

120

125

A147

I130

I27

130

N137 A57

11

15N (p.p.m.)

78

10

9

8

7

6

Fig. 2. (1H, 15N) HSQC experiments of (U-13C, 15N) CaM. (a) CaM synthesized by the E. coli cell-free system. (b) CaM synthesized with the N-terminal tag following its removal by thrombin digestion. The extra peaks in (a) and the peaks in (b) are labeled with their assignments (reproduced from ref. 14 with permission from Elsevier Science).

5

Cell-Free Protein Production for NMR Studies

79

6. Transfer the collected supernatant to fresh DEPC-treated/ autoclaved tubes. Centrifuge at 30,000 × g for 30 min at 4°C and collect ~1.0 mL of the supernatant per gram of E. coli and place it into a 50-mL tube. 7. Incubate the 50-mL tube at 37°C for 80 min with shaking (see Note 14). 8. Dialyze the solution for 45 min against 2 L of S30 buffer at 4°C using dialysis tubing with a MWCO of 6–8,000 kDa. Repeat the dialysis, and then centrifuge the solution at 15,000 × g for 10 min at 4°C. 9. Apply the supernatant to a gel-filtration column preequilibrated at 4°C with S30 buffer. Load the supernatant onto the column. Maintain a continuous flow of S30 buffer in the column. When the sample first begins to exit the column, as judged from its color and turbidity (see Note 15), collect, in bulk, 1.4 times the total volume applied to the column. 10. Dialyze the collected bulk solution at 4°C for 40–50 min against 700 mL of an equal weight mixture of PEG-8000 and S30 buffer (see Note 16). Adjust the dialysis time so as to concentrate the extract to 0.86 times the initial volume. Dialyze the extract against 2 L of S30 buffer at 4°C for 60 min to remove PEG-8000. 11. Transfer the dialyzed S30 extract to 1.5-mL tubes, freeze it in liquid nitrogen, and store it at −80°C (see Note 17). 12. Assess the extent to which the endogenous amino acids in the extract are eliminated by preparing a protein by using an 2H-, 13 C-, and 15N-labeled amino acid mixture, e.g., SAIL amino acids set. The remaining endogenous amino acids give rise to proton signals in 13C-filtered 1H NMR experiments, as shown in Fig. 3. 3.2. Preparation of T7 RNA Polymerase

1. Using a glycerol stock of E. coli strain BL21 (DE3) transformed with a plasmid encoding T7 RNA polymerase, inoculate 10 mL of LB medium containing the appropriate antibiotic in a 50-mL tube and then incubate with shaking at 37°C overnight. 2. Inoculate 1 L of M9 medium supplemented with tryptone peptone, containing the appropriate antibiotic, with the 10 mL culture from step 1 into a 2-L flask that has been prewarmed to 37°C. Incubate the cells with shaking at 37°C until the culture reaches an OD600 of 0.5. 3. Induce the expression of T7 RNA polymerase by adding IPTG to a final concentration of 0.5 mM. Incubate the cells with shaking for 8 h at 37°C and then centrifuge the culture at 5,000 × g for 10 min at 4°C. Store the cell pellet at −80°C.

80

M. Takeda and M. Kainosho

Fig. 3. 13C-filtered 1H-NMR spectra of CaM. (a) 15N-labeled CaM synthesized with the conventional extract. (b) 2H-, 13C-, 15N-labeled CaM synthesized with the conventional S30 extract. (c) 2H-, 13C-, 15N-labeled CaM synthesized with the improved, dialyzed S30 extract. All of the spectra are adjusted for the intensities of the amide region. Since the peaks of the protons attached to 13C are filtered, only the protons attached to 12C give rise to resonances in the aliphatic region (reproduced from ref. 14 with permission from Elsevier Science).

4. Suspend the frozen cell pellet in 72 mL of sonication buffer A. Add 18 mL of sonication buffer B and stir the mixture for 15 min at 4°C (see Note 18). Add 7.5 mL of 0.8% sodium deoxycholate and stir the mixture for 15 min at 4°C. Disrupt the cells with an ultrasonic generator (five rounds of 1-min sonication at 5-min intervals). 5. Add 15 mL of 2 M ammonium sulfate and bring the volume of the lysate to 150 mL by adding sonication buffer A. Then, gradually add 15 mL of 10% polyethyleneimine and stir the mixture on ice for 20 min. Centrifuge at 39,000 × g for 15 min at 4°C.

5

Cell-Free Protein Production for NMR Studies

81

6. Gradually add 0.82 volume of saturated ammonium sulfate to the supernatant. Stir on ice for 15 min and centrifuge the mixture at 12,000 × g for 15 min at 4°C. 7. Resuspend the precipitate in 45 mL of dialysis buffer containing 100 mM NaCl and 20 μg/mL PMSF. Dialyze the suspension against 2 L of dialysis buffer containing 100 mM NaCl and 20 μg/mL PMSF for 3 h using a membrane with a molecular weight cutoff of 6,000–8,000. Repeat the dialysis for 3 h and overnight. 8. Centrifuge the dialysate at 12,000 × g for 10 min at 4°C. Dilute the dialysate with one volume of dialysis buffer containing 20 μg/mL of PMSF to produce conductivity equal to that of the dialysis buffer plus 50 mM NaCl. Load the dialysate on an SP-Sepharose FF column (3 × 30 cm), equilibrated with dialysis buffer containing 50 mM NaCl and 20 μg/mL of PMSF, and run the 2-column volumes of dialysis buffer containing 50 mM NaCl and 20 μg/mL of PMSF at a flow rate of 1 mL/min to elute impurities. If necessary, to further remove impurities remaining in the column, load up to 500 mL of dialysis buffer containing 50 mM NaCl and 20 μg/mL PMSF at a flow rate of 2 mL/min (see Note 19). 9. Elute the T7 RNA polymerase with elution buffer and dialyze it against dialysis buffer containing 100 mM NaCl and 20 μg/mL of PMSF at 4°C overnight. 10. Repeat the dialysis for 6 h (see Note 20). Finally, dialyze the T7 RNA polymerase against dialysis buffer containing 100 mM NaCl and 50% glycerol at 4°C overnight. Centrifuge at 18,000 × g for 10 min at 4°C, collect the supernatant, and determine the concentration of the T7 RNA polymerase by measuring the optical density at 280 nm (ε280 = 1.4 × 105 M−1 cm−1) (see Note 21). Transfer the protein to 1.5-mL tubes and store it at −20°C. 3.3. Cell-Free Synthesis Reaction

1. Wear sanitary gloves (see Note 22) and thaw the LM mixture and the S30 extract (see Note 23). 2. Prepare the reaction and dialysis solutions (Subheading 2.3). In the case of a small-scale reaction for evaluating expression levels, the typical volumes of the reaction and dialysis solutions are 0.5 and 2.0 mL, respectively. For large production quantities, each volume is scaled up while maintaining the volume ratio of the reaction solution to the dialysis solution (see Note 24). 3. Transfer the dialysis solution to an RNase-free vessel. Tie one end of the dialysis tube firmly. Transfer the reaction solution to the dialysis tube, and tie off the other end. Fold the tubing 4–6 times and place it into the vessel such that the dialysis tube is completely submerged in the dialysis solution.

82

M. Takeda and M. Kainosho

4. Incubate the vessel with shaking for 4–8 h at 37°C (see Note 25). 5. Retrieve the reaction and dialysis solutions. If the expressed protein has a molecular weight smaller than molecular weight cutoff of the membrane, then it will be found in both the reaction and dialysis solutions. 6. To evaluate the expression level, centrifuge 50-μL aliquots of the reaction and dialysis solutions at 5,000 × g for 1 min and 4°C for. Mix the supernatant with an equal volume of 2× loading buffer, incubate for 5 min at 95°C, and load 10 μL on a polyacrylamide gel. Dissolve the precipitate in 50 μL of phosphate buffer (pH 7.0), mix it with an equal volume of 2× loading buffer, and load 10 μL on a polyacrylamide gel.

4. Notes 1. Water, treated with DEPC and then autoclaved, can also be used as RNase-free water (19). However, handle it carefully, since DEPC is a carcinogen. 2. The expression level of BL21 star is 1.2–1.4 times higher than that of the A19 strain. 3. The disruption of the E. coli cells can be accomplished by using equipment other than a French press. 4. The uniformity of the filled resin affects the resolution of gel filtration. The resin is used only once for each preparation. 5. While the use of PEG increases the expression level in some cases, it hampers SDS-PAGE analysis. Therefore, PEG should be removed by ethanol precipitation when the sample is subjected to SDS-PAGE analysis. 6. The prepared LM mixture can be frozen at −20°C for 1 month or more. 7. The creatine phosphate and creatine kinase should be prepared just prior to use. 8. If 15N labeling of the side-chain amide groups in asparagine and glutamine residues is intended, use (15N)-NH4OAc. 9. While we prepare the T7 RNA polymerase ourselves, commercially available T7 RNA polymerase can also be used. 10. The cell-free system uses a large amount of the plasmid, and thus a high-copy plasmid saves both cost and time. 11. The growth rate of the cells in the culture correlates with the activity of the resulting extract (20). We strongly recommend monitoring the growth rate. If the grow rate is slow, then it

5

Cell-Free Protein Production for NMR Studies

83

might be better to restart the culture. On the other hand, overgrowth, i.e., OD600 larger than 0.9, leads to reduced activity of the cells. 12. Do not allow the suspension to foam. 13. The cell disruption is a critical step that affects the activity of the extract. 14. This runoff reaction was performed with a preincubation mixture (14). The expression level does not change, even without the supplement (21). 15. The fraction to be collected looks yellow. 16. Before use, stir the PEG-S30 buffer at 4°C with a large magnetic stir bar to avoid PEG deposition. 17. The presence of DTT is necessary for preserving the activity over time. The activity of an S30 extract prepared without DTT decreases by 10–20% after 3 months of storage at −80°C. 18. The viscosity of the solution increases because of the lysozyme. 19. Wash until the optical density at 280 nm becomes constant. 20. The protein may precipitate during the dialysis. 21. If necessary, concentrate the solution up to about 10 mg/mL with an ultrafiltration device. 22. When setting up the cell-free reaction, use sanitary gloves to prevent contamination with RNases. 23. The frozen S30 extract should be thawed gently on ice. 24. Tryptophan and tyrosine residues are unlikely to be soluble. If needed, warm the amino acid solutions to 60°C. Do not vigorously shake the solution after the template DNA has been added. 25. PEG is assumed to play a role as a stabilizer of the system. For example, in the case of CaM, the ratio of protein yields with and without PEG was 1.6. However, even a small amount of PEG can disturb the SDS-PAGE quantitation of the production level of a target protein. If the solution contains PEG, then precipitate the supernatant with ethanol. Empirically, however, it is difficult to completely remove PEG even by ethanol precipitation. Therefore, optimizing the PEG concentration is somewhat difficult in small-scale cell-free reactions.

Acknowledgments This work was supported by a grant from the Targeted Protein Research Program (MEXT) to M. Kainosho and a Grant-in-Aid for Young Scientists (B) (21770110) to M. Takeda.

84

M. Takeda and M. Kainosho

References 1. Clemens, M.J., and Prujin, G.J. (1999) Protein synthesis in Eukaryotic Cell-free systems. Oxford University Press, New York, pp. 129–165. 2. Kramer, G., Kudlicki, W., and Hardesty, B. (1999) Cell-free Coupled TranscriptionTranslation systems from Escherichia coli. Oxford University Press, New York, pp. 129–165. 3. Zubay, G. (1973) In vitro synthesis of protein in microbial systems. Ann. Rev. Gen. 7, 267–287. 4. Spirin, A.S., Barano, V.I., Ryabova, L.A., Ovodov, S.Y., and Alakhov, Y.B. (1988) A continuous cell-free translation system capable of producing polypeptides in high yield. Science 242, 1162–1164. 5. Kim, D.M., Kigawa, T., Choi, C.Y., and Yokoyama, S. (1996) A highly efficient cell-free protein synthesis system from Escherichia coli. Eur. J. Biochem. 239, 881–886. 6. Kim, D.M., and Swartz, J.R. (2000) Prolonging cell-free protein synthesis by selective reagent additions. Biotechnol. Prog. 16, 385–390. 7. Kigawa, T., Yabuki, T., Yoshida, Y., Tsutsui, M., Ito, Y., Shibata, T., and Yokoyama, S. (1999) Cell-free production and stable-isotope labeling of milligram quantities of proteins. FEBS Lett. 442,15-19. 8. Madin, K., Sawasaki, T., Ogasawara, T., and Endo, Y. (2000) A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomes. Proc. Natl. Acad. Sci. U.S.A. 97, 559–564. 9. Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., and Ueda, T. (2001) Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755. 10. Kigawa, T., Muto, Y., and Yokoyama, S. (1995) Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis. J. Biomol. NMR 6, 129–134. 11. Kainosho, M., Torizawa, T., Iwashita, Y., Terauchi, T., Ono. A,M., and Güntert, P. (2006) Optimal isotope labelling for NMR

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

protein structure determinations. Nature 440, 52–57. Takeda, M., Ikeya, T., Güntert, P., and Kainosho, M. (2007) Automated structure determination of proteins with the SAIL-FLYA NMR method. Nature Protocol 2, 2896–2902. Kainosho, M., and Güntert, P. (2010) SAILStereo-array isotope labeling. Q. Rev. Biophys. 7, 1–54. Torizawa, T., Shimizu, M., Taoka, M., Miyano, H., and Kainosho, M. (2004) Efficient production of isotopically labeled proteins by cell-free synthesis: A practical protocol. J. Biomol. NMR 30, 311–325. Pratt, J.M. (1984) Transcription and Translation: A Practical Approach, IRL Press, New York, pp. 179–209. Davanloo, P., Rosenberg, A.H., Dunn, J.J., and Studier, F.W. (1984) Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 81, 2035–2039. Zawadzki, V., and Gross, H.J. (1991) Rapid and simple purification of T7 RNA polymerase. Nucl. Acid Res. 19,1948. Grodberg, J., and Dunn, J.J. (1988) ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J. Bacteriol. 170, 1245–1253. Huang, Y.H., Leblanc, P., Apostolou, V., Stewart, B., and Moreland, R.B. (1995) Comparison of Milli-Q PF Plus water to DEPC-treated water in the preparation and analysis of RNA. Biotechniques 19, 656–661. Zawada, J., and Swartz, J.R. (2006) Effects of growth rate on cell extract performance in cellfree protein synthesis. Biotechnol. Bioeng. 94, 618–624. Liu, D.V., Zawada J.F., and Swartz, J.R. (2005) Streamlining Escherichia coli S30 extract preparation for economical cellfree protein synthesis. Biotechnol. Prog. 21, 460–465.

Chapter 6 Cell-Free Membrane Protein Expression for Solid-State NMR Alaa Abdine, Kyu-Ho Park, and Dror E. Warschawski Abstract Although cell-free expression is a relative newcomer to the biochemical toolbox, it has already been reviewed extensively, even in the more specialized cases such as membrane protein expression, nanolipoprotein particles, and applications to crystallography and nuclear magnetic resonance (NMR). Solid-state NMR is also a newcomer to the structural biology toolbox, with its own specificities in terms of sample preparation. Cell-free expression and solid-state NMR are a promising combination that has already proven useful for the structural study of membrane proteins in their native environment, the hydrated lipid bilayer. We describe below several protocols for preparing MscL, a mechanosensitive membrane channel, using cell-free expression destined for a solid-state NMR study. These protocols are flexible and can easily be applied to other membrane proteins, with minor adjustments. Key words: In vitro synthesis, Integral membrane proteins, Membrane protein reconstitution, Liposomes, Solid-state NMR

1. Introduction Cell-free expression is one of the major new developments in structural biology, both for nuclear magnetic resonance (NMR) and X-ray crystallography, because it allows for overproduction of proteins, both wild-type and mutant, that are often produced with prohibitively low yields using classical biosynthetic methods. Proteins that are expressed cell-free end up in the reaction container and hence in water where membrane proteins precipitate, often irreversibly. Cell-free expression was first thought to be impractical for membrane protein expression because the system did not include a biological apparatus for targeting the protein to the membrane (1). Providing the medium with detergent was also considered risky since detergent could perturb protein expression by interfering with the ribosome, polymerase or any other essential

Alexander Shekhtman and David S. Burz (eds.), Protein NMR Techniques, Methods in Molecular Biology, vol. 831, DOI 10.1007/978-1-61779-480-3_6, © Springer Science+Business Media, LLC 2012

85

86

A. Abdine et al.

component of the cell-free expression system. These obstacles were overcome in 2004, when several groups, using optimized cell lysates, developed protocols for membrane protein expression in vitro, in the presence of detergents (2–4). For X-ray crystallography and solution-state NMR, the major techniques used in structural biology today, membrane protein structure determination is still a challenge: first, because the aforementioned necessity of obtaining large quantity of functional proteins is aggravated, in vivo, by the limited membrane surface available (5) and second, because membrane proteins are hydrophobic and have to be manipulated in detergents at all times when they are extracted from their native environment, the lipid bilayer (6). Detergents or other surfactants are used for membrane solubilization and during protein purification (which often requires an affinity tag, such as a polyhistidine stretch). They can interfere with protein crystallization or make aggregates that are too large for solution-state NMR. Last but not the least, detergents can interfere with protein folding or function, and the protein structure or dynamics determined in a detergent environment is not necessarily representative of the native structure or dynamics (7, 8). Nevertheless, since 1985, over 200 membrane protein structures have been determined by using X-ray crystallography (9) and, since 1997, about 30 structures by using solution-state NMR (10). In this context, membrane protein cell-free expression has been developed and reviewed, especially for isotope labeling strategies, another issue for NMR, where in vitro synthesis offers a very efficient and versatile alternative that greatly minimizes amino acid scrambling (11, 12). Solid-state NMR is an alternative for membrane protein structure determination inside the hydrated lipid bilayer, where the protein is correctly folded and stable for a long time. Sample preparation for solid-state NMR is therefore different than for solutionstate NMR. If the protein is expressed cell-free in the presence of detergent, it needs to be purified, renatured and reconstituted in a membrane bilayer. Compared to a protein expressed in a cell, the solubilization step is avoided and the purification step is greatly simplified and accelerated, reducing the chances of spurious proteolysis. Reconstitution of the protein in a lipid membrane is the price to pay to obtain a sample where the membrane protein is almost certainly in its native state, and where the protein function can be checked and monitored (13, 14). All these steps are quite flexible, and they are described below for the expression of the mechanosensitive channel MscL. This protocol is general for membrane proteins, although details such as the nature or the concentration of detergent can vary from one protein to the next. Cell-free expression in presence of liposomes or nanodiscs is an alternative sample preparation approach that has been developed more recently and that has yet to be proven general for membrane

6

Cell-Free Membrane Protein Expression for Solid-State NMR

87

proteins (15–17). When possible, it presents many advantages for solid-state NMR studies. First and foremost, the protein is already in its final state, saving time and avoiding many biochemical steps where the protein may be partially lost or inactivated. Second, the protein is purified simply by centrifugation, alleviating the necessity of adding an affinity tag. In addition, no detergent is used that can interfere and that needs to be removed. Importantly, the protein is in its native environment at all times, where it is well folded, functional and stable (18). Finally, this approach is quite flexible, allowing for a variety of labeling strategies (19). In our hands, cell-free expression of MscL in the presence of liposomes has proven efficient (3, 14, 18, 19) and we describe it below, as it is the best method so far for providing a solid-state NMR sample. The protocols presented here make use of commercial kits for cell-free expression, which we found advantageous for its convenience, reliability, for saving the time and manpower to make the lysate, and also for managing the stocks. Cell lysates can also be prepared, following published protocols from various cell types such as bacteria, wheat germ, or others (11, 20). We describe below an optimized procedure for expressing the membrane protein MscL, using the commercial Roche/5Prime continuous exchange vessel and kits, with an Escherichia coli extract. Since, for each sample preparation protocol, it is necessary to check for protein integrity, we also describe several tests that should be performed to assess the quality of the sample.

2. Materials 2.1. Cell-Free Expression of the Mechanosensitive Channel MscL in Detergent Micelles

All solutions are prepared with autoclaved nuclease-free Milli-Q water (see Note 1). 1. Thermoregulated shaker for the cell-free reaction vessel. 2. Nuclease-free 50-mL tubes. 3. Nuclease-free 1.5-mL tubes. 4. Nuclease-free glass pipettes. 5. Nuclease-free pipette tips (0–10, 10–200, 200–1,000 μL, and 1–5 mL), autoclaved at 121°C for 20 min. 6. pIVEX-2.3-mscL plasmid (3). Encodes the E. coli MscL C-terminally fused to a His6 tag under the control of the T7 promoter. Dissolve in pure nuclease-free water and store aliquoted at −20°C, at 0.5 μg/μL. 7. RTS 9000 cell-free expression kit (Roche/5Prime). Contains lyophilized E. coli lysate, reaction mix, feeding mix, reconstitution buffer, continuous exchange reaction vessel, and a syringe (see Fig. 1, Notes 2–5). Store at −80°C.

88

A. Abdine et al.

Fig. 1. Roche/5Prime RTS 9000 reaction vessel. The reaction compartment (10 mL) and the feeding compartment (100 mL) are each accessible through two screws. The reaction compartment contains the cell lysate, plasmid DNA, detergent or preformed liposomes and is where the coupled transcription/translation reaction takes place. The separate feeding compartment provides additional ions, energy substrates, nucleotides and amino acids, through a semipermeable membrane (MW cutoff 10 kDa). Simultaneously, by-products that may inhibit the reaction are diluted through the same membrane into the feeding compartment. This continuous exchange allows cell-free expression to last for up to 24 h.

8. Unlabeled amino acids (powder), store at −20°C. 9.

13

C/15N labeled amino acids (powder), store at −20°C.

10. Dithiothreitol (DTT): Prepare 40 mM stock, store at −20°C. 11. 20% (w/v) Triton X-100: Prepare 200 mL, stir until homogeneous, store at 4°C for up to 6 months. 12. Unlabeled amino acid solutions: Prepare 168 mM solutions of each amino acid except for leucine (140 mM) and the labeled amino acids, Ile and Thr, with the appropriate solution (see Note 6). The amount of amino acid to be incorporated is calculated in Subheading 3.1 and is indicated in Table 1. Transfer the appropriate volume of each unlabeled amino acid to a 1.5-mL vial, and sonicate until the solution looks clear. Transfer the individual solutions to a 50-mL tube. Add Tyr and Leu last to avoid precipitation; prepare fresh before using and keep on ice. 13. Labeled amino acid solutions, Ile and Thr: Dissolve 23 mg of Ile and 3.9 mg of Thr in 1.0 and 0.20 mL of reconstitution buffer, respectively (~168 mM each), into 1.5-mL tubes; prepare fresh before using and keep on ice (see Subheading 3.1, Table 1 and Note 7). 14. FPLC purification system. 15. 1 M NaOH: To adjust pH, store at room temperature. 16. 100 mM NiSO4: Store at room temperature. 17. 20% Ethanol: Store at room temperature for up to a month. 18. 4-(2-Aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF): 100 mM stock solution in water, store at 4°C for up to 6 months.

6

Cell-Free Membrane Protein Expression for Solid-State NMR

89

Table 1 Preparation of amino acid solutions for MscL cell-free expression in a volume V = 110 mL (see Subheading 3.1 and Fig. 1) Amino acid

MWi (g/mol)

n

Amount (mg)

Volume (mL)

Ala (A)

89

15

15

0.98

Arg (R)

174

6

11

0.39

Asn (N)

132

6

Asp (D)

133

7

10

0.46

Cys (C)

121

0

0

0.10

Gln (Q)

146

4

6.4

0.26

Glu (E)

147

8

13

0.52

Gly (G)

75

13

11

0.85

His (H)

155

7

12

0.46

Ile (I)

131

16

23

1.0

Leu (L)

131

13

19

1.0

Lys (K)

146

9

14

0.59

Met (M)

149

5

Phe (F)

165

10

Pro (P)

115

7

8.9

0.46

Ser (S)

105

4

4.6

0.26

Thr (T)

119

3

3.9

0.20

Trp (W)

204

0

0

0.10

Tyr (Y)

181

1

2.0

0.10

Val (V)

117

13

8.7

8.2 18

17

0.39

0.33 0.65

0.85

n is the number of each amino acid type, of molecular weight MWi, in the protein sequence. The weight of each amino acid is (n × MWi × 1.1 × 10−2), expressed in mg. Since each amino acid is solubilized at 168 mM, except for leucine, at 140 mM, the corresponding volume of each amino acid solution is (n × 11)/168, except for leucine where it is (n × 11)/140, expressed in mL

19. Chelating column (5 mL): Stored in 20% ethanol at 4°C. 20. FPLC buffer A1: 50 mM NaH2PO4–NaOH (7.1 g/L), pH 8, 300 mM NaCl (17.5 g/L), 10 mM imidazole (680 mg/L), 4.0% Triton X-100 (40 g/L), store at 4°C. 21. FPLC buffer A2: 50 mM NaH2PO4–NaOH (7.1 g/L), pH 8, 300 mM NaCl (17.5 g/L), 10 mM imidazole (680 mg/L), 0.2% Triton X-100 (2 g/L), store at 4°C.

90

A. Abdine et al.

22. FPLC buffer B: 50 mM NaH2PO4–NaOH (7.1 g/L), pH 8, 300 mM NaCl (17.5 g/L), 500 mM imidazole (34 g/L), 0.2% Triton X-100 (2 g/L), store at 4°C. 23. FPLC sample loading buffer: 50 mM NaH2PO4–NaOH (7.1 g/L), pH 8, 300 mM NaCl (17.5 g/L), 10 mM imidazole (680 mg/L), 1% Triton X-100 (10 g/L), store at 4°C. 24. 10× Dialysis buffer: 0.1 M HEPES–KOH, pH 7.5, 1 M KCl, and 2% (w/v) Triton X-100, store at 4°C for up to 6 months. 25. 1× Dialysis buffer: Prepare 2 L by diluting 200 mL of 10× buffer in 1,750 mL of water, adjusting the pH to 7.5 using 4 M KOH, and adding water to 2 L final volume. Store at 4°C for up to 6 months. 26. 2 dialysis cassettes with a 10 kDa MW cutoff. 27. Methanol. 28. DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine powder, store at −20°C. 29. 4 M KOH: To adjust pH, store at room temperature. 30. 0.8% Triton X-100: Prepare 10 mL, dissolve and stir until homogeneous, store at 4°C for up to 6 months. 31. Wet polystyrene beads: Wash 5 g of 300–1,200 μm polystyrene beads with 25 mL of pure methanol and then wash four times with 25 mL of Milli-Q water. The beads are kept in water at 4°C and are stable for months, provided a weekly renewal of Milli-Q water. 32. HEPES–KCl buffer: 10 mM HEPES–KOH, pH 7.5, 100 mM KCl, store at 4°C for up to 6 months. 33. HEPES solutions for solubilizing Tyr, Trp, and Phe (Subheading 2.1, see Notes 6 and 7): 60 mM HEPES, pH 13 for Tyr, pH 1 for Trp, and pH 7.5 for Phe. Adjust the pH using KOH or HCl. Keep on ice. 2.2. Cell-Free Expression of the Mechanosensitive Channel MscL in Liposomes

All solutions are prepared with autoclaved nuclease-free Milli-Q water (see Note 1). 1. Thermoregulated shaker for the cell-free reaction vessel. 2. Mini-Extruder with two 1-mL gastight microsyringes and 0.1-μm cutoff polycarbonate membranes. 3. Nuclease-free 50-mL tubes. 4. Nuclease-free 1.5-mL tubes. 5. Nuclease-free glass pipettes. 6. Nuclease-free pipette tips (0–10, 10–200, 200–1000 µL and 1–5 mL). Autoclave at 121°C for 20 min.

6

Cell-Free Membrane Protein Expression for Solid-State NMR

91

7. pIVEX-2.3-mscL plasmid: For protein expression in liposomes, an affinity tag such as a polyhistidine stretch is preferable but not mandatory. 8. RTS 9000 cell-free expression kit (Roche/5Prime). Contains lyophilized E. coli lysate, reaction mix, feeding mix, reconstitution buffer, a continuous exchange reaction vessel, and a syringe (see Fig. 1, Notes 2–5), store at −80°C. 9. Unlabeled amino acids (powder), store at −20°C. 10.

13

C/15N labeled amino acids (powder), store at −20°C.

11. DTT (dithiothreitol): Prepare 40 mM stock, store at −20°C. 12. 4 M KOH: To adjust pH, store at room temperature. 13. AEBSF (Subheading 2.1). 14. DOPC. 15. Chloroform. 16. HEPES–KCl buffer (Subheading 2.1). 17. DOPC liposome preparation: Prepare a chloroform solution of 10 mg/mL of DOPC (100 mg of DOPC in 10 mL of dry chloroform). Dry the lipids and then resuspend with RTS reconstitution buffer to obtain a 20 mg/mL aqueous solution. Sonicate 2.5 mL of the solution for 5 min, and then extrude it 13 times with a 0.1-μm filter, to obtain a liposome solution containing 40–50 mg of lipids, and store at 4°C. This is the amount of lipids that is expected in the final sample. DOPC can be replaced by other lipids or lipid mixtures, at a higher or lower concentration. 18. HEPES solutions for solubilizing Tyr, Trp, and Phe (Subheading 2.1). 19. Unlabeled amino acid solutions: Prepare 168 mM solutions of each amino acid except for leucine (140 mM) and the labeled amino acids (item 20), with the appropriate solution (see Note 6). The amount of amino acid to be incorporated is calculated in Subheading 3.1 and is indicated in Table 1. The appropriate volume of each unlabeled amino acid is transferred to a 1.5-mL vial, and sonicated until the solution looks clear. The vials are transferred into a 50-mL tube. Tyr and Leu are added last, to avoid precipitation; prepare fresh before using and keep on ice. 20. Labeled amino acid solutions, Arg, Ile, Pro, Met, and Phe: Weigh 11 mg of Arg, 23 mg of Ile, and 8.9 mg of Pro and dissolve in 0.39, 1.0, and 0.46 mL of reconstitution buffer, respectively (~168 mM). Weigh 8.2 mg of Met and dissolve in 0.33 mL of reconstitution buffer plus 50 μL of 40 mM DTT. Weigh 18 mg of Phe and dissolve in 0.65 mL of the appropriate HEPES solution (Subheading 2.1), and sonicate for 1 min. Prepare each amino acid solution fresh before using and keep on ice (see Subheading 3.1, Table 1 and Note 7).

92

A. Abdine et al.

2.3. Sample Characterization

All solutions described should be prepared using Milli-Q water and stored at room temperature unless stated otherwise. 1. FA diluted solution: 37% formaldehyde in water, store at 4°C. 2. Dimethyl sulfoxide (DMSO). 3. 1 M NaOH: To adjust pH. 4. 1 M HCl: To adjust pH. 5. 1 M Tris base, pH 8. 6. SDS running buffer: 100 mM Tris base, 100 mM HEPES, 0.1% SDS, adjust to pH 8 using NaOH or HCl. 7. Staining buffer: 0.5% Coomassie Brilliant Blue R-250 dye, 50% ethanol and 10% acetic acid. 8. Destaining buffer: 20% ethanol and 10% acetic acid. 9. 2× SDS loading buffer: 0.2 M Tris base, pH 8, 8% SDS, 40% glycerol, 0.4% bromophenol blue, 0.4 M DTT, adjust pH 8 using 1 M NaOH or 1 M HCl. 10. 1× SDS loading buffer: dilute 1 mL of 2× SDS loading buffer with 1 mL of water. 11. DSS solution: 50 mM disuccinimidylsuberate in DMSO. 12. HEPES–KCl buffer (see Subheading 2.1). 13. Polyvinylidene fluoride (PVDF) membranes. 14. TBS-Tween buffer 1: 50 mM Tris base, pH 7.4, 150 mM NaCl, 0.05% Tween-20. 15. Monoclonal anti-histidineperoxidase-conjugate antibody solution (for the dot blot): Dilute 2,000× from the stock solution with TBS-Tween buffer 1, prepare fresh. 16. Diaminobenzidine solution: Dissolve one tablet of diaminobenzidine and one tablet of urea hydrogen peroxide in 5 mL of water, prepare fresh. 17. Western-blotting detection kit. 18. Nonfat dry milk (powder). 19. Chemiluminescence reagents (H2O2 and luminol), store at 4°C. 20. Towbin buffer: 25 mM Tris base, 192 mM glycine, 20% isopropanol, and 0.1% SDS. Store without isopropanol at 4°C, add isopropanol just before use. 21. Ponceau S solution: 0.1% Ponceau S, 5% acetic acid. 22. TBS-Tween buffer 2: TBS-Tween buffer 1 with 5% (w/v) nonfat dry milk, freshly made. 23. Mouse anti-histidine antibody solution (for the Western-blot): Dilute 5,000× from the stock solution with TBS-Tween buffer 1, prepare fresh. 24. Anti-mouseperoxidase-coupled antibody solution (for the Western-blot): Dilute 10,000× from the stock solution with TBS-tween buffer 1, prepare fresh.

6

Cell-Free Membrane Protein Expression for Solid-State NMR

93

25. Micro BCA Protein Assay kit (Pierce). 26. Nuclease-free 1.5-mL tubes. 27. Bovine serum albumine (BSA): 1 mg/mL stock solution. 28. Solubilization buffer: 0.2% Triton X-100, solubilized in HEPES–KCl buffer, store at 4°C for up to 6 months. 29. Acetone, stored at −20°C. 30. Nitric acid solution: 1%, store at room temperature in a safety cabinet. Handle under the hood. 31. Perchloric acid solution: 70%, store at room temperature in a safety cabinet. Handle under the hood. 32. Ammonium molybdate solution: Dissolve 2.5 g in 100 mL of water, store at 4°C. 33. Ascorbic acid solution: Dissolve 10 g in 100 mL of water, prepare fresh, and do not keep for more than 1 week at 4°C. 34. Phosphate stock solution: Dissolve 10 mg of KH2PO4 in 100 mL of water and store at 4°C. 35. Nuclease-free 3-mL ultracentrifugation tubes. 36. Sucrose. 37. Sucrose solutions: 10, 20, and 45% (w/v) sucrose, solubilized in HEPES–KCl buffer, store at 4°C. 38. Nitrocellulose membrane (for Western blotting). 39. X-ray film (to develop the proteoliposomes must be mixed with sucrose Western blot). 40. Film developer. 2.4. NMR Sample Preparation

Four millimeter diameter rotors for high-resolution magic-angle spinning solid-state NMR (see Fig. 2).

Fig. 2. Four millimeter diameter rotors for high-resolution magic-angle spinning solidstate NMR. The sample (approximately 50 μL) is contained in the center of the zirconium rotor, thanks to the Teflon insert bottom and top, and is kept tightly in place by the top screw and the Kel-F cap.

94

A. Abdine et al.

3. Methods The methods outlined below describe the following: (1) calculation of the amount of incorporated amino acids, (2) cell-free expression of the mechanosensitive channel MscL in detergent micelles, purification of protein/detergent micelles, protein reconstitution into liposomes, (3) cell-free expression directly into liposomes, (4) sample characterization, and (5) NMR sample preparation. 3.1. Cell-Free Expression of the Mechanosensitive Channel MscL in Detergent Micelles

The method below describes the production of an MscL sample for solid-state NMR, using a cell-free protein expression system in detergent micelles. Although any labeling scheme could be performed, the sample described below was labeled on isoleucines and threonines, using 13C/15N-labeled amino acids, and was subsequently used in solid-state NMR experiments (14).

3.1.1. Day 1: Cell-Free Protein Expression in Detergent Micelles

1. Calculate the amount of amino acids to be incorporated during protein expression. The concentration of each amino acid in the protein cell-free expression system (V = 110 mL, see Fig. 1) is usually ~1 mM. For a given protein, each amino acid concentration depends on its occurrence, n, in the protein sequence. In our case, we have obtained good results with a final concentration of each amino acid equal to n/10, expressed in mM. The corresponding weight of each amino acid, of molecular weight MWi, is therefore (n × V × MWi/10), expressed in mg. The corresponding volume of each amino acid solution of concentration Ci, where Ci is 168 or 140 μM (Subheading 2.1), is (n × V/10)/Ci, expressed in mL. The weights and final volumes calculated for this protocol are summarized in Table 1. 2. Thaw the DTT, RTS reconstitution buffer, and MscL plasmid at room temperature. 3. Thaw the other components of the RTS kit (E. coli lysate, reaction mix, and feeding mix) on ice. 4. Prepare the unlabeled amino acid solutions (see Subheading 2.1). 5. Prepare the labeled amino acid solutions (see Subheading 2.1). 6. Add the labeled amino acid solution to the unlabeled amino acid solution. 7. Add 3 mL of 40 mM DTT, sonicate (80 W for 5 min or until clear), and store on ice. 8. Reconstitute the lyophilized E. coli lysate in 5.2 mL of reconstitution buffer. Shake the bottle gently (see Note 8). 9. Reconstitute the lyophilized reaction mix in 2.2 mL of reconstitution buffer. Shake the bottle gently (see Note 8).

6

Cell-Free Membrane Protein Expression for Solid-State NMR

95

10. Reconstitute the lyophilized feeding mix in 80 mL of reconstitution buffer. Shake the bottle gently (see Note 8). 11. Prepare the feeding solution by adding 26 mL of the reconstituted amino acid solutions (mixture of unlabeled and labeled amino acid solutions) and 3 mL of 40 mM DTT to the feeding mix (80 mL). 12. Prepare the reaction solution by adding the reconstituted reaction mix (2.2 mL), 2.7 mL of the reconstituted amino acid solution and 0.3 mL of 40 mM DTT to the reconstituted E. coli lysate (5.2 mL). Remove a 20 μL aliquot of this solution for later comparison on gel electrophoresis. 13. Add 300 μL of the MscL plasmid (~150 μg) to the reaction solution. 14. Add 200 μL of 20% Triton X-100 solution to the reaction solution. 15. Add 2.2 mL of 20% Triton X-100 solution to the feeding solution. 16. Open both screws of the reaction compartment and fill the reaction compartment with the reaction solution using a nuclease-free pipette (see Fig. 1). Remove air bubbles by tapping the vessel lightly. 17. Open both screws of the feeding compartment and fill the feeding compartment with the feeding solution, using the provided syringe (see Fig. 1). Remove air bubbles by tapping the vessel lightly. 18. Insert the reaction vessel into a thermoregulated shaker. 19. Set the shaking speed to 800 rpm. 20. Set the temperature to 30°C. 21. Incubate the reaction for 22 h. 3.1.2. Day 2: Purification of Detergent/Protein Micelles

1. After 22 h, stop the cell-free protein expression. Remove a 20 μL aliquot of the reaction run-on solution for gel electrophoresis. 2. Remove the reaction run-on mix using a pipette, add 100 μL of the AEBSF solution to prevent protease digestion, and stir at room temperature for 15 min (see Note 9). 3. Dilute the sample to a final volume of 50 mL using FPLC buffer A1 and centrifuge at 10,000 × g for 15 min at 4°C. 4. Connect the chelating column to the FPLC system (see Note 10). Pass 4–5 column volumes (CV) of distilled water through the column, to wash away the ethanol solution in which it has been kept. 5. Degas the FPLC buffers for 10 min in an ultrasonic bath. Wash the pump inlets with the degassed solutions. Wash the system with FPLC buffer A1 until the UV and conductivity baselines are stable.

96

A. Abdine et al.

6. At the end of the sample centrifugation (step 3), dilute the supernatant (reaction run-on mix) with FPLC sample loading buffer to a final volume of 100 mL (final dilution ~10×). 7. Filter this solution through a 0.45-μm filter. 8. Resuspend the pellet with 5 mL of FPLC buffer A1 and remove a 20 μL aliquot for gel electrophoresis. 9. Introduce half (50 mL) of the diluted reaction run-on mix into the column (see Note 11). Collect the flow-through in the first 50-mL tube. 10. Wash off the nonspecifically bound material by raising the concentration of imidazole to 100 mM, by mixing 80% of FPLC buffer A1 with 20% of FPLC buffer B (6 CV total). The concentration of detergent remains high (around 3%). Collect the flow-through in the second 50-mL tube. 11. To elute the protein, switch FPLC buffer A1 to buffer A2, to reduce the detergent concentration to 0.2%. Collect the flowthrough in the second 50-mL tube. 12. Increase the imidazole concentration to 200 mM by mixing 60% of solution A2 with 40% of solution B (2 CV total), and collect eluant fractions of 2 mL each (see Note 12). 13. Increase the imidazole concentration to 500 mM by switching to 100% FPLC buffer B (6 CV total) and collect eluant fractions of 2 mL each. 14. Wash the column with FPLC buffer A1 (10 CV or until the UV and conductivity baselines are stable), then repeat steps 9–13 with the remaining 50 mL (second half of the diluted reaction run-on mix, see Note 11). 15. At the end of the FPLC protein purification run (see Note 10), the potentially interesting fractions are separated into approximately 40 tubes, whereas the flow-though is contained in two 50-mL tubes. Remove a 20 μL aliquot from each of these 42 tubes, for gel electrophoresis. 16. Determine which fractions contain the histidine-tagged protein by performing a dot blot (see Subheading 3.3.4). 17. Wash the two dialysis cassettes in 1× dialysis buffer. 18. For each half of the solution, pool the fractions containing the protein into a dialysis cassette. 19. Dialyze against 1 L of 1× dialysis buffer, overnight at 4°C. 3.1.3. Day 3: Sample Characterization and Reconstitution into Liposomes

1. Dialyze a second time against 1 L of 1× dialysis buffer for 2 h at 4°C. Remove a 20 μL aliquot for gel electrophoresis. 2. Assess the cell-free expression level, FPLC purification efficiency, and oligomeric state by SDS-PAGE (see Subheadings 3.3.1 and 3.3.3).

6

Cell-Free Membrane Protein Expression for Solid-State NMR

97

3. After SDS-PAGE, wash the gel in water and perform a Westernblot transfer (see Subheading 3.3.5). 4. Estimate the protein concentration. Since MscL does not contain any tryptophan it has to be quantified by using the BCA method (see Subheading 3.3.6 and Note 13). 5. BCA quantification of our MscL preparation indicated that each half of the solution contains approximately 4.8 mg of protein in a 10 mL volume that will be reconstituted in 19.2 mg of DOPC liposomes (protein–lipid ratio of 1:4). DOPC can be replaced by other lipids or lipid mixtures, at a higher or lower concentration (21). Lipids can also be replaced by other mixtures to reconstitute the protein into bicelles or nanodiscs (22, 23). 6. Split the solutions into four fractions of 5 mL, each containing 2.4 mg of protein and 10 mg of Triton X-100. To each fraction, add 9.6 mg of DOPC solubilized in 2.5 mL of the 0.8% Triton X-100 solution, such that the final lipid–detergent ratio is 1:3. Stir slowly for 15 min at room temperature. 7. To each fraction of 7.5 mL add 300 mg of wet polystyrene beads (detergent–bead ratio is 1:10) and put the four tubes on a rotator, to remove the detergent by adsorption onto the beads. Incubate overnight at 4°C. For alternative methods (21, 24), see Note 14. 3.1.4. Day 4: Preparation of the Sample for Solid-State NMR

1. Add another 300 mg of wet polystyrene beads to each tube. Incubate for another 2 h, at 4°C. 2. After 2 h, set the tubes vertically for 5 min and discard the pellet and the beads. 3. Divide the supernatant into four tubes and centrifuge at 100,000 × g for 30 min at 4°C. Remove a 20 μL aliquot of one supernatant for gel electrophoresis, discard the rest and resuspend the pellets with 1 mL of HEPES–KCl buffer for each pellet. Split the new resuspended pellets into two tubes and centrifuge at 100,000 × g for 30 min at 4°C. Repeat one more time so that the entire sample fits into a single tube. Remove a final 20 μL aliquot of the supernatant for gel electrophoresis. Dry the final pellet under argon and store it at −20°C. 4. Assess the reconstitution by SDS-PAGE (see Subheading 3.3.1), and by proteoliposome density characterization on a sucrose gradient (see Subheading 3.3.9). Also, estimate the lipid and water content (see Subheadings 3.3.8 and 3.3.10, and Note 15). 5. The average quantity of protein obtained with the RTS 9000 kit is 10 mg in 10 mL of reaction mix, which is generally sufficient to prepare two or three NMR samples. Transfer ~30 mg of pelleted sample to the NMR rotor (see Subheading 3.4). 6. Store the remaining pellet at −20°C.

98

A. Abdine et al.

3.2. Cell-Free Expression of the Mechanosensitive Channel MscL in Liposomes

The method below describes the production of an MscL sample for solid-state NMR, using a cell-free protein expression system directly into lipid vesicles. Although any labeling scheme could be performed, the sample described below was labeled on arginines, isoleucines, methionines, phenylalanines and prolines, using 13 C/15N-labeled amino acids, and was subsequently used in solidstate NMR experiments (19). Lipids used were synthetic DOPC, but other lipids or complex mixtures can also be used, such as asolectin (18) or nanodiscs (17).

3.2.1. Day 1: Cell-Free Protein Expression

1. Thaw the DTT solution, RTS reconstitution buffer and MscL plasmid at room temperature. 2. Thaw the other components of the RTS kit (E. coli lysate, reaction mix, and feeding mix) on ice. 3. Prepare the DOPC liposomes (Subheading 2.2). 4. Prepare the HEPES solutions (Subheading 2.1). 5. Prepare the unlabeled amino acid solutions (Subheading 2.1). 6. Prepare the labeled amino acid solutions (Subheading 2.1). 7. Add the labeled amino acid solutions to the unlabeled amino acid solutions. 8. Add 3 mL of 40 mM DTT, sonicate (80 W for 5 min or until clear), and store on ice. 9. Reconstitute the lyophilized E. coli lysate in 2.7 mL of reconstitution buffer. Shake the bottle gently (see Note 8). 10. Add the liposome solution (2.5 mL) to the E. coli lysate once the latter is completely dissolved. 11. Reconstitute the lyophilized reaction mix in 2.2 mL of reconstitution buffer. Shake the bottle gently (see Note 8). 12. Reconstitute the lyophilized feeding mix in 80 mL of reconstitution buffer. Shake the bottle gently (see Note 8). 13. Prepare the feeding solution by adding 26 mL of the reconstituted amino acid solution (mixture of unlabeled and labeled amino acid solutions) and 3 mL of 40 mM DTT to the feeding mix solution (80 mL). 14. Prepare the reaction solution by adding the reconstituted reaction mix (2.2 mL), 2.7 mL of the reconstituted amino acid solution and 0.3 mL of 40 mM DTT to the reconstituted E. coli lysate with the liposomes (5.2 mL). Take a 20 μL aliquot of this reaction solution for later comparison on gel electrophoresis. 15. Add 300 μL of the MscL plasmid (~150 μg) to the reaction solution. 16. Open both screws of the reaction compartment and fill the reaction compartment with the reaction solution using a nuclease-free pipette (see Fig. 1). Remove any air bubbles by tapping the vessel lightly.

6

Cell-Free Membrane Protein Expression for Solid-State NMR

99

17. Open both screws of the feeding compartment and fill the feeding compartment with the feeding solution, using the provided syringe (see Fig. 1). Remove any air bubbles by tapping the vessel lightly. 18. Insert the reaction vessel into a thermoregulated shaker. 19. Set the shaking speed to 800 rpm. 20. Set the temperature to 30°C. 21. Incubate the reaction for 22 h. 3.2.2. Day 2: Preparation of the Sample for Solid-State NMR

1. After 22 h, stop the cell-free protein expression. Remove a 20 μL aliquot of the reaction run-on solution for gel electrophoresis. 2. Remove the reaction run-on mix using a pipette, add 100 μL of the AEBSF solution to prevent protease digestion, and stir at room temperature for 15 min (see Note 9). 3. Split the sample into six tubes and centrifuge at 100,000 × g for 30 min at 4°C. Remove a 20 μL aliquot of one supernatant for gel electrophoresis, discard the rest and resuspend each pellet with 1 mL of HEPES–KCl buffer. Split the resuspended pellets into three tubes and centrifuge at 100,000 × g for 30 min at 4°C. Repeat one more time so that the entire sample fits into a single tube. Remove a final 20 μL aliquot of the supernatant for gel electrophoresis. Dry the final pellet under argon and store it at −20°C. 4. Assess cell-free expression efficiency and oligomeric state by SDS-PAGE (see Subheadings 3.3.2 and 3.3.3). At this point, the expressed protein is generally almost pure. 5. If the protein was expressed with a polyhistidine tag, after SDSPAGE, wash the gel in water and perform a Western-blot transfer (see Subheading 3.3.5). 6. Estimate the protein and lipid concentration using the BCA and Rouser methods respectively (see Subheadings 3.3.7 and 3.3.8, and Note 13). 7. Characterize the proteoliposome density on a sucrose gradient (see Subheading 3.3.9), and estimate the water content (see Subheading 3.3.10 and Note 15). 8. The average quantity of protein obtained with the RTS 9000 kit is 10 mg in 10 mL of reaction mix, which is generally sufficient to prepare two or three NMR samples. About 30 mg of pelleted sample is transferred into the NMR rotor (see Subheading 3.4). 9. Store the remaining pellet at −20°C.

100

A. Abdine et al.

3.3. Sample Characterization 3.3.1. Electrophoresis of Protein Expressed in Detergent Micelles

1. Perform SDS-PAGE on the following samples: (a) Aliquots from the reaction mix before and after the cell-free expression reaction, to check for expression of the target protein. (b) Aliquots from the centrifugation pellet and supernatant, to check whether the protein was totally solubilized in detergent micelles or if there are some aggregates. (c) Aliquots from the purification flow-through and the purified fractions. (d) Analyze the incorporation of the protein by comparing aliquots of the protein-detergent micelles with the proteoliposomes, after the reconstitution step. (e) Aliquots of the supernatant of all the wash steps. (f) Also check the integrity of the purified protein after dialysis. 2. Dilute all SDS PAGE samples 1:1 with 2× SDS loading buffer. 3. Heat the samples to 90°C for 5 min. 4. Cool the samples and load on a gel. 5. Run the gel using SDS running buffer. 6. Wash the gel in water and stain/destain the gel using staining/ destaining buffers, respectively.

3.3.2. Electrophoresis of Protein Expressed in Liposomes

1. Perform SDS-PAGE on the following samples: (a) Aliquots from the reaction mix before and after the cellfree expression reaction, to check for expression of the target protein. (b) Also check the centrifugation supernatants. 2. Follow steps 2–6 in Subheading 3.3.1.

3.3.3. Electrophoresis for Oligomeric State Characterization

If the protein is an oligomer (like MscL), assess its integrity by cross-linking the protein with different free primary amine targeting reagents, like formaldehyde (FA) or disuccinimidylsuberate (DSS) (3, 25). NB: the protein buffer must not contain primary amines (such as in Tris). 1. Mix 5 μg of purified MscL, either in detergent or liposomes, in 10 μL of HEPES–KCl buffer and 0.54 μL of the FA diluted solution. 2. Mix 5 μg of purified MscL, either in detergent or liposomes, in 10 μL of HEPES–KCl buffer and 0.2 μL of 50 mM DSS. 3. Incubate both samples, without shaking, for 30 min at room temperature.

6

Cell-Free Membrane Protein Expression for Solid-State NMR

101

Fig. 3. Coomassie blue stained SDS-PAGE of MscL: (1) purified in detergent, (2) crosslinked with formaldehyde, (3) cross-linked with disuccinimidylsuberate. Formaldehyde generates five protein bands on the gel, corresponding to various oligomeric forms, from monomers to pentamers, while disuccinimidylsuberate generates mostly pentamers, confirming the pentameric nature of Escherichia coli MscL (3, 25).

4. Stop the cross-linking reactions by adding 2 μL of 1 M Tris base, pH 8, to each sample. Gently mix and incubate for 10 min at room temperature. 5. Add 3 μL of 2× SDS loading buffer to each sample and mix. 6. Heat the FA sample to 60°C and the DSS sample to 90°C, for 5 min in a water bath. 7. Cool the samples and load on a gel. 8. After migration, wash the gel in water and stain/destain the gel using staining/destaining buffers, respectively (see Fig. 3) or perform a Western-blot transfer (see Subheading 3.3.5). 3.3.4. Dot Blot

1. Deposit 2 μL of each fraction of purified protein directly onto a PVDF membrane. 2. Allow the protein solution to air-dry. 3. Prepare the TBS-Tween buffers 1 and 2. 4. Prepare the monocolonal antibody solution. 5. When the protein spots are completely dry, incubate the membrane in the TBS-Tween buffer 2 for 30 min. 6. Wash the membrane twice for 5 min with the TBS-Tween buffer 1.

102

A. Abdine et al.

7. Incubate the membrane with the monoclonal antibody solution for 1 h. 8. Wash the membrane twice for 5 min with the TBS-Tween buffer 1. 9. Prepare the diaminobenzidine solution. 10. Dry the membrane on a tissue. 11. Incubate the membrane with the diaminobenzidine solution for a few seconds, until the spots are colored. 12. Dry the membrane on a tissue. The color remains on the spots containing the histidine-tagged protein, while it vanishes on the other spots. 3.3.5. Semidry Western-Blot

1. Prepare the Towbin buffer. 2. Prepare the transfer sandwich by superimposing two filter papers, the gel, the nitrocellulose membrane and another two filter papers in the Towbin buffer. 3. Place the transfer sandwich on the anode plate and clip the cathode plate on top. 4. Clamp the potential at 25 V and blot for 20 min. 5. Prepare TBS-Tween buffers 1 and 2, mouse anti-histidine antibody, and anti-mouse peroxidase-coupled antibody solutions. 6. Assess the sample transfer and relative concentration by staining the membrane with Ponceau S solution. This is a reversible stain that can be removed by washing in TBS-Tween buffer 1. 7. After protein transfer and destaining, saturate the nitrocellulose membrane by incubating in TBS-Tween buffer 2 for 30 min. 8. Rinse the membrane quickly with TBS-Tween buffer 1. 9. Incubate for 45 min at room temperature with the mouse antihistidine antibodies solution. 10. Wash the membrane for 4 × 5 min with TBS-Tween buffer 1. 11. Incubate for 30 min at room temperature with the anti-mouse peroxidase-coupled antibody solution. 12. Wash the membrane for 5 × 5 min with TBS-Tween buffer 1. 13. Incubate the membrane for 1 min with 1 mL of each of the chemiluminescence reagents (H2O2 and luminol). 14. In a light tight box, expose X-ray films to the membrane for varying times, usually between 30 s and 2 min, and develop the films in the photo developer.

6 3.3.6. Protein Quantification in Detergents

Cell-Free Membrane Protein Expression for Solid-State NMR

103

Protein concentration is measured using the BCA method (26, 27), using the test tube procedure of the Micro BCA Protein Assay Kit: 1. Prepare eight BSA standards by diluting the stock solution with the solubilization buffer (the final BSA concentration should fall between 0.5 and 20 μg/mL). Make three replicates of each dilution. Introduce 500 μL of each into 1.5-mL tubes (24 tubes in total) and store at room temperature. 2. Dilute the unknown protein sample with the solubilization buffer to three different concentrations that are expected to be between 1 and 20 μg/mL. Make three replicates of each dilution. Transfer 500 μL of each sample into 1.5-mL tubes (nine tubes in total). 3. Add 500 μL of Micro BCA Working Reagent to each of the 1.5-mL tubes (24 BSA standards and nine unknown proteins) and incubate for 60 min at 60°C. Cool to room temperature. 4. Measure the absorbance at 562 nm. 5. Plot a standard curve based on the absorbance of the BSA samples. 6. Deduce the protein concentration of each unknown protein sample.

3.3.7. Protein Quantification in Proteoliposomes

Lipids need to be removed from the sample by precipitating the proteins using cold acetone, followed by a centrifugation to separate the protein pellet from the supernatant containing the lipids: 1. Prepare the 24 BSA standard tubes (Subheading 3.3.6, step 1). 2. Dilute the unknown protein sample with the solubilization buffer to three different concentrations that are expected to be between 1 and 20 μg/mL. Make three replicates of each dilution. Transfer 500 μL of each sample into 1.5-mL tubes (nine tubes in total). 3. Add 1 mL of cold acetone to each tube of unknown protein. Vortex the tubes and incubate for 60 min at −20°C. Centrifuge at 10,000 × g for 10 min at room temperature. Discard the supernatant. Incubate the tubes for 30 min at room temperature to allow for acetone evaporation. Add 500 μL of the solubilization buffer, and vortex the tubes again. 4. Add 500 μL of Micro BCA Working Reagent to each of the 1.5-mL tubes (24 BSA standards and nine unknown proteins) and incubate for 60 min at 60°C. Cool to room temperature. 5. Measure the absorbance at 562 nm. 6. Plot a standard curve based on the absorbance of the BSA samples. 7. Deduce the protein concentration of each unknown protein sample.

104

A. Abdine et al.

3.3.8. Lipid Quantification in Proteoliposomes

The phospholipid content in the proteoliposome sample is assessed by the Rouser method, which measures the phosphate concentration in the sample (28). Full eye, face, and skin protection is required for this method. 1. Set a heating block at 180°C. 2. Wash the glass tubes in nitric acid solution before use and dry them in an oven. 3. Prepare five phosphate standard samples by diluting the phosphate stock solution into five samples containing 1–5 μg of KH2PO4 per tube. Classically, 5 μg of KH2PO4 give an absorbance of 0.9 at 800 nm. Store at 4°C. 4. Prepare the ascorbic acid solution (Subheading 2.3). 5. Collect three proteoliposomes volumes containing approximately 1–5 μg of lipids. Transfer the samples into clean glass tubes. 6. Add 0.65 mL of the perchloric acid solution and place the tubes in the heated block at 180°C for 30 min of digestion, until the yellow color has disappeared. 7. Add 0.65 mL of the perchloric acid solution per phosphate standard tube (digestion is not necessary). 8. Put all the tubes on ice, and set the heated block at 100°C (alternatively, a boiling water bath can be used). 9. Once cooled, add to the tubes the following: 3.3 mL of water, 0.5 mL of the ammonium molybdate solution, and 0.5 mL of the ascorbic acid solution. Agitate on a vortex after each addition. 10. Put the tubes in the heated block at 100°C for 5 min. 11. Put the tubes on ice for 5 min. 12. Read the absorbance of the cooled samples at 800 nm. 13. Plot a standard curve based on the absorbance of the phosphate standard samples. The exact concentration in the phospholipid samples is calculated based on the standard curve.

3.3.9. Proteoliposome Density Characterization

A discontinuous sucrose flotation gradient analysis (21, 29) can be performed to check that the membrane protein is correctly reconstituted into proteoliposomes. The proteoliposomes can be layered at the bottom (see below) or at the top (see Note 16) of the sucrose layers. 1. After MscL reconstitution, add 120 mg of sucrose to 0.2 mL of the proteoliposome suspension (containing approximately 96 μg of protein) and mix gently by pipetting, until the sucrose has completely dissolved. The final volume is approximately 0.265 mL and the final sucrose concentration is 45%. Adjust the final volume to 0.4 mL with the buffered 45% sucrose solution.

6

Cell-Free Membrane Protein Expression for Solid-State NMR

105

2. Transfer the resulting suspension to a 3-mL ultracentrifuge tube, and keep it vertical in an appropriate rack for the following steps. 3. Carefully deposit 0.7 mL of the buffered 20% sucrose solution, with minimal flow along the tube wall to avoid layers mixing. 4. Repeat with 0.7 mL of the buffered 10% sucrose solution. 5. Ultracentrifuge at 100,000 × g for 1.5 h in a swinging rotor at 18°C. 6. At the end of the centrifugation, carefully remove and transfer the tube into the rack, keeping it vertical. Carefully collect six fractions of 0.3 mL, from top to bottom, into 1.5-mL tubes, and adjust the final volume to 1 mL with water. 7. Wash the bottom of the 3 mL tube with 100 μL of 1× SDS loading buffer and remove a 15 μL aliquot for gel electrophoresis. 8. Centrifuge the 1.5-mL tubes at 16,000 × g for 15 min at room temperature, to pellet the proteoliposomes and remove the sucrose. 9. Resuspend each pellet in 100 μL of 1× SDS loading buffer and take a 15 μL aliquot of each for gel electrophoresis. 10. Analyze the aliquots by gel electrophoresis. For an expected ratio of protein to lipid of 1:4 (w/w), proteoliposomes appear at the interface between 20% and 10% sucrose. Protein-free liposomes lie at the top of the sucrose gradient, while lipid-free protein aggregates lie at the bottom of the sucrose gradient (see Note 17). 3.3.10. Water Quantification in the Sample

3.4. NMR Sample Preparation

Water content is estimated by weighing 10 mg of sample prior to and after 16 h in vacuum. 1. Weigh the empty rotor, cap, insert bottom, top, and screw (see Fig. 2). 2. Introduce the insert bottom into the rotor. 3. Introduce a couple of mg of the pelleted sample into the rotor on the tip of a spatula. Place the rotor into a 1.5-mL tube and centrifuge it at 10,000 × g for 1 min at room temperature. Repeat this until approximately 30 mg of sample has been introduced into the rotor. 4. Place the insert top into the rotor, without the top screw. Clean the upper part the rotor with a precision wiper before capping tightly. At this point, the rotor containing sample should be stored at −20°C until the NMR experiment is performed. 5. Introduce the rotor into the magic-angle spinning NMR probe and into the magnet of the NMR spectrometer. Spin the rotor at ~10 kHz for 15 min.

106

A. Abdine et al.

6. Extract the rotor and open it. Clean the upper part of the rotor again, in case a drop of water has come out. Place the top screw, tighten it, and then cap the rotor tightly. Weigh the full rotor to deduce the final sample mass. A typical sample consists of 3 mg proteins, 12 mg lipids and 15 mg water.

4. Notes 1. All solutions require Milli-Q water, but only solutions used inside the cell-free expression vessel need to be autoclaved and nuclease-free, to make sure that no ribonucleases are present that would damage the RNAs in the lysate. 2. Continuous-exchange cell-free protein expression with an RTS commercial kit from Roche/5Prime is advantageous for its convenience, reliability, for saving time and manpower in preparing the lysate, and for managing the stocks. The thermoregulated shaker, on the contrary, is not necessarily from Roche/5Prime. 3. Cell-free reactions should be performed on a small scale (RTS 100 or analog kits) for optimization studies, including detergents or lipids. Protein yield should be evaluated in a cell-free kit equipped with a continuous-exchange system (e.g., RTS 500), where the yield is usually higher. Once the protocol is optimized, the proteins can then be expressed on larger scales (e.g., RTS 9000). 4. Cell-free kits were stored at −80°C rather than −20°C, which increases the lifespan of the kits to over a year. Freeze–thaw cycles should be avoided. 5. Circular DNA was used for cell-free expression of MscL. Linear PCR templates can also be used, but they require larger amounts of DNA. DNA quality is important for cell-free expression. A plasmid purification procedure with a good yield (typically 100 μg of plasmid DNA per standard MIDI-prep), including an anion-exchange step, was found to be necessary. DNA should be dissolved in either nuclease-free water (our case) or Tris solution (typically 10 mM, at a pH between 8 and 8.5), but not in a buffer containing EDTA, as it would change the free magnesium ion concentration and reduce the protein expression yield. 6. Some amino acids are difficult to solubilize. Sonication or heating at 60°C can improve solubility. Met (M) and Cys (C) have to be supplemented with DTT (4 and 8 mM respectively). Tyr (Y), Trp (W), and Phe (F) have to be solubilized in 60 mM HEPES in nuclease-free water, adjusting the pH to 13, 1, or

6

Cell-Free Membrane Protein Expression for Solid-State NMR

107

7.5 respectively, using KOH or HCl. However, Trp (W), Asp (D), Asn (N), Cys (C), or Tyr (Y) will not dissolve completely and have to be used as suspensions. Alternatively, the Roche/5Prime RTS Amino acid Sampler provides appropriate stock solutions of each individual unlabeled amino acid. 7. Commercial mixtures of labeled amino-acids are also available (30). All labeled amino-acids can be specifically incorporated into the expressed proteins, except for Gln (Q) and Glu (E) when using the Roche/5Prime buffer, which contains large quantities of unlabeled glutamate. Some scrambling is observed with Ser (S), Asp (D), Asn (N), Gln (Q), and Glu (E). Some metabolic degradation can occur with Arg (R), Cys (C), Trp (W), Met (M), Asp (D), and Glu (E) during prolonged incubation. In such a case, an increased amount of amino acids can enhance the protein production. 8. Too vigorous mixing should be avoided while preparing the different lysates, as it may denature proteins or ribosomes in the extracts. 9. AEBSF is added after the expression is complete to prevent protease degradation of the target product. Proteases inhibitors can also be added to the reaction chamber, but they should be tested on a small scale beforehand. 10. If the chelating column is not precharged with Ni ions, before connecting it to the FPLC system, open it at both ends and wash it with 2 CV of distilled water, 1 CV of 100 mM EDTA, pH 8, 3 CV of distilled water, 2 CV of 0.1 M NiSO4 solution, and 5 CV of distilled water. Once the column is charged, an additional washing step with 5 CV of 20% ethanol is necessary before storage at 4°C. At the end of the protein purification run, wash the column with water (5 CV), 0.5 M NaOH (5 CV), water (5 CV) and store in 20% ethanol. 11. The total reaction run-on is expected to contain approximately 10 mg of protein but also detergent and impurities, which may bind to the column as well. Since the binding saturation of the column surface is on the order of 10 mg/mL, the reaction run-on is split in two so as to always remain below the saturation limit. 12. For MscL, the fractions containing the protein are eluted between 200 and 500 mM of imidazole, but this is highly protein dependent. Depending on the histidine-tag accessibility, the protein may elute at lower or higher imidazole concentrations. 13. Protein concentration is usually estimated using UV fluorescence of aromatic amino acids. If the protein is not sufficiently fluorescent, quantification can be performed using the commercial Micro BCA bicinchonic acid protein assay kit (Pierce). Care should be taken if lipids are present in the sample.

108

A. Abdine et al.

14. Instead of using polystyrene beads, detergent extraction can be performed using cyclodextrin inclusion compounds, as described by DeGrip et al. (24), or by dialysis (21). 15. Proteoliposomes can be dehydrated and rehydrated, but care should be taken, since the process may affect protein activity. 16. The discontinuous sucrose flotation gradient analysis to check that the membrane protein is correctly reconstituted can be performed with the proteoliposomes layered either at the top or at the bottom of the sucrose layers, or both (21, 29). If at the top, the proteoliposomes must be mixed with sucrose for a final sucrose concentration of 10%. The rest of the protocol is the same: After layering 0.7 mL of 45% sucrose, then 0.7 mL of 20% sucrose and then 0.7 mL of the proteoliposomes in 10% sucrose, the proteoliposomes usually appear at the interface between 20 and 10% sucrose. 17. If the amount of lipid-free protein aggregates is not negligible, the discontinuous sucrose flotation gradient can also be used as a purification technique by layering the entire reconstituted proteoliposome suspension.

Acknowledgments This work was supported by fellowships from the Ministère de l’Enseignement Supérieur et de la Recherche and the Fondation pour la Recherche Médicale (to A.A.), by the CNRS (UMR 7099 and 8619), the ANR (ANR-06-JCJC0014), the Univ Paris Diderot and the Université Paris-Sud 11. We thank Alexandre Ghazi, Catherine Berrier, Emmanuelle Billon-Denis, and Michiel A. Verhoeven for helping us optimize the protocols presented here. References 1. Lyford, L. K., and Rosenberg, R. L. (1999) Cell-free expression and functional reconstitution of Homo-oligomeric α7 Nicotinic Acetylcholine Receptors into Planar Lipid Bilayers. J. Biol. Chem. 274, 25675–25681. 2. Elbaz, Y., Steiner-Mordoch, S., Danieli, T., and Schuldiner, S. (2004) In vitro synthesis of fully functional EmrE, a multidrug transporter, and study of its oligomeric state. Proc. Natl. Acad. Sci. U.S.A. 101, 1519–1524. 3. Berrier, C., Park, K. H., Abes, S., Bibonne, A., Betton, J. M., and Ghazi, A. (2004) Cell-free synthesis of a functional ion channel in the absence of a membrane and in the presence of detergent. Biochemistry 43, 12585–12591.

4. Klammt, C., Lohr, F., Schafer, B., Haase, W., Dötsch, V., Ruterjans, H., Glaubitz, C., and Bernhard, F. (2004) High level cell-free expression and specific labeling of integral membrane proteins. Eur. J. Biochem. 271, 568–580. 5. Wagner, S., Bader, M. L., Drew, D., and de Gier, J. W. (2006) Rationalizing membrane protein overexpression. Trends Biotechnol. 24, 364–371. 6. Eshaghi, S. (2009) High-throughput expression and detergent screening of integral membrane proteins. Methods Mol. Biol. 498, 265–271. 7. Tate, C. G. (2010) Practical considerations of membrane protein instability during purification and crystallisation. Methods Mol. Biol. 601, 187–203.

6

Cell-Free Membrane Protein Expression for Solid-State NMR

8. Breyton, C., Pucci, B., and Popot, J.-L. (2010) Amphipols and fluorinated surfactants: Two alternatives to detergents for studying membrane proteins in vitro. Methods Mol. Biol. 601, 219–245. 9. White, S. H. (2010) Membrane Proteins of Known Structure. University of California at Irvine. http://blanco.biomol.uci.edu/ Membrane_Proteins_xtal.html Accessed 25 July 2011. 10. Warschawski, D. E. (2010) Membrane Proteins of Known Structure Determined by NMR. Drorlist. http://www.drorlist.com/nmr/MPNMR.html Accessed 25 July 2011. 11. Schneider, B., Junge, F., Shirokov, V. A., Durst, F., Schwarz, D., Dötsch, V., and Bernhard, F. (2010) Membrane protein expression in cellfree systems. Methods Mol. Biol. 601, 165–186. 12. Sobhanifar, S., Reckel, S., Junge, F., Schwarz, D., Kai, L., Karbyshev, M., Löhr, F., Bernhard, F., and Dötsch, V. (2010) Cell-free expression and stable isotope labelling strategies for membrane proteins. J. Biomol. NMR 46, 33–43. 13. Lehner, I., Basting, D., Meyer, B., Haase, W., Manolikas, T., Kaiser, C., Karas, M., and Glaubitz, C. (2008) The key residue for substrate transport (Glu14) in the EmrE dimer is asymmetric. J. Biol. Chem. 283, 3281–3288. 14. Abdine, A., Verhoeven, M. A., Park, K.-H., Ghazi, A., Guittet, E., Berrier, C., Van Heijenoort, C., and Warschawski, D. E. (2010) Structural study of the membrane protein MscL using cell-free expression and solid-state NMR. J. Magn. Reson. 204, 155–159. 15. Kalmbach, R., Chizhov, I., Schumacher, M. C., Friedrich, T., Bamberg, E., and Engelhard, M. (2007) Functional cell-free synthesis of a seven helix membrane protein: in situ insertion of bacteriorhodopsin into liposomes. J. Mol. Biol. 371, 639–648. 16. Marques, B., Liguori, L., Paclet, M. H., VillegasMendéz, A., Rothe, R., Morel, F., Lenormand, J.-L. (2007) Liposome-mediated cellular delivery of active gp91(phox). PLoS One 2, e856. 17. Katzen, F., Fletcher, J. E., Yang, J. P., Kang, D., Peterson, T. C., Cappuccio, J. A., Blanchette, C. D., Sulchek, T., Chromy, B. A., Hoeprich, P. D., Coleman, M. A., and Kudlicki, W. (2008) Insertion of membrane proteins into discoidal membranes using a cell-free protein expression approach. J. Proteome Res. 7, 3535–3542. 18. Berrier, C., Guilvout, I., Bayan, N., Park, K.-H., Mesneau, A., Chami, M., Pugsley, A. P., and Ghazi, A. (2011) Coupled cell-free synthesis and lipid vesicle insertion of a functional oligomeric channel MscL - MscL does not need the insertase YidC for insertion in vitro. Biochim. Biophys. Acta 1808, 41–46.

109

19. Abdine, A., Verhoeven, M. A., and Warschawski, D. E. (2011) Cell-free expression and labeling strategies for a new decade in solid-state NMR. New Biotechnol. 28, 272–276. 20. He, M. (2008) Cell-free protein synthesis: applications in proteomics and biotechnology. New Biotechnol. 25, 126–132. 21. Rigaud, J.-L., and Lévy, D. (2003) Reconstitution of Membrane Proteins into Liposomes. Methods Enzymol. 372, 65–86. 22. Triba, M. N., Zoonens, M., Popot, J.-L., Devaux, P. F., and Warschawski, D. E. (2006) Reconstitution and alignment by a magnetic field of a β-barrel membrane protein in bicelles. Eur. Biophys. J. 35, 268–275. 23. Leitz, A. J., Bayburt, T. H., Barnakov, A. N., Springer, B. A., and Sligar, S. G. (2006) Functional reconstitution of β2-adrenergic receptors utilizing self-assembling Nanodisc technology. BioTechniques 40, 601–612. 24. De Grip, W. J., Van Oostrum, J., and BoveeGeurts, P. H. M. (1998) Selective detergentextraction from mixed detergent/lipid/protein micelles, using cyclodextrin inclusion compounds: a novel generic approach for the preparation of proteoliposomes. Biochem. J. 330, 667–674. 25. Sukharev, S. I., Schroeder, M. J., and McCaslin, D. R. (1999) Stoichiometry of the Large Conductance Bacterial Mechanosensitive Channel of E. coli. A Biochemical Study. J. Membrane Biol. 171, 183–193. 26. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85. 27. Wiechelman, K. J., Braun, R. D., and Fitzpatrick, J. D. (1988) Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation. Anal. Biochem. 175, 231–237. 28. Rouser, G., Fkeischer, S., and Yamamoto, A. (1970) Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 5, 494–496. 29. Laird, D. M., Eble, K. S., and Cunningham, C. C. (1986) Reconstitution of mitochondrial F0.F1-ATPase with phosphatidylcholine using the nonionic detergent, octylglucoside. J. Biol. Chem. 261, 14844–14850. 30. Etezady-Esfarjani, T., Hiller, S., Villalba, C., and Wüthrich, K. (2007) Cell-free protein synthesis of perdeuterated proteins for NMR studies. J. Biomol. NMR 39, 229–238.

Chapter 7 Expression and Purification of Src-family Kinases for Solution NMR Studies Andrea Piserchio, David Cowburn, and Ranajeet Ghose Abstract NMR analyses of the structure, dynamics, and interactions of the Src family kinases (SFKs) have been hindered by the limited ability to obtain sufficient amounts of properly folded, soluble protein from bacterial expression systems, to allow these studies to be performed in an economically viable manner. In this chapter, we detail our attempts to overcome these difficulties using the catalytic domain (SrcCD) of c-Src, the prototypical SFK, as an illustrative example. We describe in detail two general methods to express and purify SrcCD from Escherichia coli expression systems in both fully active wild-type and kinase-deficient mutant forms, allowing the efficient and cost-effective labeling by NMR-active isotopes for solution NMR studies. Key words: Protein tyrosine kinases, Src-family kinases, Escherichia coli expression systems

1. Introduction Protein kinases in higher eukaryotes transfer the γ-phosphate of ATP to the hydroxyl groups of specific serine, threonine (serine/ threonine kinases) or tyrosine (tyrosine kinases) residues in substrate proteins. This covalent modification of the Ser/Thr/ Tyr–OH moiety is a fundamental mechanism of cellular regulation and intracellular signal transduction (1–3) and has a significant role in almost every aspect of cell growth, differentiation, maturation, motility, and regulated cell death. In vivo, the phosphorylation levels of kinase targets are tightly controlled by the opposing actions of kinases and their corresponding phosphatases (4, 5). A loss of this control due to mutations, changes in expression levels, alteration of protein–protein interactions including localization, and/or catalytic activity has been implicated in a variety of diseases

Alexander Shekhtman and David S. Burz (eds.), Protein NMR Techniques, Methods in Molecular Biology, vol. 831, DOI 10.1007/978-1-61779-480-3_7, © Springer Science+Business Media, LLC 2012

111

112

A. Piserchio et al.

including a multitude of human cancers (6–9), making protein kinases significant and established targets for anticancer therapies (10–13). Eukaryotic protein kinases contain a highly conserved catalytic domain (CD) (14, 15) that forms a dual-lobed structure (Fig. 1) with a smaller, mainly β-sheet N-terminal lobe (N-lobe), and a larger, mainly α-helical C-terminal lobe (C-lobe). The catalytic activity, while wholly contained within the CD, is often regulated by additional modular domains (16) or insertions within the CD itself (17). While crystallographic analyses of both serine/threonine (18) and tyrosine kinases (19) have provided valuable insights into the regulation of their catalytic activity, several questions remain. The current hypothesis is that most eukaryotic protein kinases function as simple two-state switches transitioning between well-defined “active” and “inactive” conformations based on the phosphorylation state of a small number of regulatory residues. Mounting evidence suggests that this may be an oversimplification and the boundary between fully active and fully inactive states may be not that well defined. Further, the simple phosphorylation-dependent switch scenario is complicated by mounting experimental evidence that hints towards the role played by longrange allosteric dynamic pathways in the regulation of kinase activity, where active-like conformations may be attained through remote interactions (20). Structural details on the nature of recognition of downstream substrates and of regulating kinases and phosphatases are still poorly defined for many of the subfamilies of

Fig. 1. (a) Domain arrangement in inactive full-length c-Src (PDB ID: 2SRC). The residues comprising the linker between the SH2 and the catalytic domain (CD), which engages the SH3 domain, is shown in ball-and-stick representation, as are the residues that comprise the C-terminal tail. The regulatory tyrosine residues in the activation loop (Y416, unphosphorylated in inactive c-Src) and the C-terminal tail (Y527, phosphorylated in inactive c-Src) are also shown in ball-and-stick representation. The ATP-analog, ANP (phosphoaminophosphonic acid-adenylate), bound to the ATP-binding site, is also shown in ball-and-stick representation. (b) Expanded view of SrcCD with the N-lobe and C-lobes indicated. Important regulatory elements that include the activation loop, Gly-rich loop, the HRD (that precedes the catalytic loop) and DFG motifs are also indicated. Y416 is shown in a ball-and-stick representation and the αC helix is indicated by a dashed ellipse. The ligand ANP is also shown bound to the ATP-binding pocket.

7

Expression and Purification of Src-family Kinases for Solution NMR Studies

113

the human kinome. This is not unexpected, given that some of these interactions may be weak or transient, and the resultant complexes may not be amenable to crystallographic analyses. Given this background, it would seem that solution NMR spectroscopy is tailor-made to resolve these unanswered issues about structure, dynamics, and interactions involving protein kinases. Targeting eukaryotic protein kinases by using solution NMR methodologies is especially important because of considerable interest in the pharmaceutical industry in designing small molecule inhibitors of enzymatic activity (21). In particular, the rational design of inhibitors targeting remote sites exerting allosteric control over catalytic activity in protein kinases (type III inhibitors) holds promise as a strategy for inhibiting kinase signaling in a highly selective fashion. These allosteric sites, which are more likely to be unique to specific protein kinases or kinase subfamilies, could be identified by NMR studies performed on a representative subgroup of the human kinome. These studies are expected to assume greater importance as the role of yet uncharacterized protein kinases in cancer becomes clearer by using a combination of RNAi technologies in tumo