Protein Purification Protocols

Cutler_FM_Final 1 10/29/03, 2:43 PM METHODS IN MOLECULAR BIOLOGY™

John M. Walker, SERIES EDITOR

268. Public Health Microbiology: Methods and Protocols, 241. Cell Cycle Checkpoint Control Protocols, edited by edited by John F. T. Spencer and Alicia L. Ragout de Howard B. Lieberman, 2004 Spencer, 2004 240. Mammalian Artificial Chromosomes: Methods and 267. Recombinant Gene Expression: Reviews and Protocols, Protocols, edited by Vittorio Sgaramella and Sandro Second Edition, edited by Paulina Balbas and Argelia Eridani, 2003 Johnson, 2004 239. Cell Migration in Inflammation and Immunity: 266. Genomics, Proteomics, and Clinical Bacteriology: Methods and Protocols, edited by Daniele D’Ambrosio Methods and Reviews, edited by Neil Woodford and and Francesco Sinigaglia, 2003 Alan Johnson, 2004 238. Biopolymer Methods in Tissue Engineering, edited 265. RNA Interference, Editing, and Modification: Methods by Anthony P. Hollander and Paul V. Hatton, 2003 and Protocols, edited by Jonatha M. Gott, 2004 237. G Protein Signaling: Methods and Protocols, edited 264. Protein Arrays: Methods and Protocols, edited by by Alan V. Smrcka, 2003 Eric Fung, 2004 236. Plant Functional Genomics: Methods and Protocols, 263. Flow Cytometry, Second Edition, edited by Teresa edited by Erich Grotewold, 2003 S. Hawley and Robert G. Hawley, 2004 235. E. coli Plasmid Vectors: Methods and Applications, 262. Genetic Recombination Protocols, edited by Alan S. edited by Nicola Casali and Andrew Preston, 2003 Waldman, 2004 234. p53 Protocols, edited by Sumitra Deb and Swati Palit 261. Protein–Protein Interactions: Methods and Applications, Deb, 2003 edited by Haian Fu, 2004 233. Protein Kinase C Protocols, edited by Alexandra C. 260. Mobile Genetic Elements: Protocols and Genomic Newton, 2003 Applications, edited by Wolfgang J. Miller and Pierre 232.Protein Misfolding and Disease: Principles and Capy, 2004 Protocols, edited by Peter Bross and Niels Gregersen, 259. Receptor Signal Transduction Protocols, Second 2003 Edition, edited by Gary B. Willars and R. A. John 231. Directed Evolution Library Creation: Methods and Challiss, 2004 Protocols, edited by Frances H. Arnold and George 258. Gene Expression Profiling: Methods and Protocols, Georgiou, 2003 edited by Richard A. Shimkets, 2004 230. Directed Enzyme Evolution: Screening and Selection 257. mRNA Processing and Metabolism: Methods and Methods, edited by Frances H. Arnold and George Protocols, edited by Daniel R. Schoenberg, 2004 Georgiou, 2003 256. Bacterial Artifical Chromosomes, Volume 2: 229. Lentivirus Gene Engineering Protocols, edited by Functional Studies, edited by Shaying Zhao and Maurizio Federico, 2003 Marvin Stodolsky, 2004 228. Membrane Protein Protocols: Expression, Purification, 255. Bacterial Artifical Chromosomes, Volume 1: Library and Characterization, edited by Barry S. Selinsky, Construction, Physical Mapping, and Sequencing, 2003 edited by Shaying Zhao and Marvin Stodolsky, 2004 227. Membrane Transporters: Methods and Protocols, 254. Germ Cell Protocols, Volume 2: Molecular Embryo edited by Qing Yan, 2003 Analysis, Live Imaging, Transgenesis, and Cloning, 226. PCR Protocols, Second Edition, edited by John M. S. edited by Heide Schatten, 2004 Bartlett and David Stirling, 2003 253. Germ Cell Protocols, Volume 1: Sperm and Oocyte 225. Inflammation Protocols, edited by Paul G. Winyard Analysis, edited by Heide Schatten, 2004 and Derek A. Willoughby, 2003 252. Ribozymes and siRNA Protocols, Second Edition, 224. Functional Genomics: Methods and Protocols, edited edited by Mouldy Sioud, 2004 by Michael J. Brownstein and Arkady B. Khodursky, 251. HPLC of Peptides and Proteins: Methods and Protocols, 2003 edited by Marie-Isabel Aguilar, 2004 223. Tumor Suppressor Genes: Volume 2: Regulation, 250. MAP Kinase Signaling Protocols, edited by Rony Function, and Medicinal Applications, edited by Wafik Seger, 2004 S. El-Deiry, 2003 249. Cytokine Protocols, edited by Marc De Ley, 2004 222. Tumor Suppressor Genes: Volume 1: Pathways and 248. Antibody Engineering: Methods and Protocols, Isolation Strategies, edited by Wafik S. El-Deiry, 2003 edited by Benny K. C. Lo, 2004 221. Generation of cDNA Libraries: Methods and Protocols, 247. Drosophila Cytogenetics Protocols, edited by Daryl edited by Shao-Yao Ying, 2003 S. Henderson, 2004 220. Cancer Cytogenetics: Methods and Protocols, edited 246. Gene Delivery to Mammalian Cells: Volume 2: Viral by John Swansbury, 2003 Gene Transfer Techniques, edited by William C. 219. Cardiac Cell and Gene Transfer: Principles, Protocols, Heiser, 2004 and Applications, edited by Joseph M. Metzger, 2003 245. Gene Delivery to Mammalian Cells: Volume 1: 218. Cancer Cell Signaling: Methods and Protocols, edited Nonviral Gene Transfer Techniques, edited by William by David M. Terrian, 2003 C. Heiser, 2004 217. Neurogenetics: Methods and Protocols, edited by 244. Protein Purification Protocols, Second Edition, Nicholas T. Potter, 2003 edited by Paul Cutler, 2004 216. PCR Detection of Microbial Pathogens: Methods 243. Chiral Separations: Methods and Protocols, edited by and Protocols, edited by Konrad Sachse and Joachim Gerald Gübitz and Martin G. Schmid, 2004 Frey, 2003 242. Atomic Force Microscopy: Biomedical Methods and 215. Cytokines and Colony Stimulating Factors: Methods Applications, edited by Pier Carlo Braga and Davide and Protocols, edited by Dieter Körholz and Wieland Ricci, 2004 Kiess, 2003

Cutler_FM_Final 2 10/29/03, 2:43 PM METHODS IN MOLECULAR BIOLOGY™

Protein Purification Protocols

Second Edition

Edited by Paul Cutler Genomic and Proteomic Sciences, Medicines Research Center, GlaxoSmithKline Pharmaceuticals, Stevenage, UK

Cutler_FM_Final 3 10/29/03, 2:43 PM © 2004 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512

www.humanapress.com

All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Biology™ is a trademark of The Humana Press Inc.

All papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher.

This publication is printed on acid-free paper. ∞ ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials.

Production Editor: Wendy S. Kopf.

Cover illustration:Paul Culter.

Cover design by Patricia F. Cleary.

For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel.: 973-256-1699; Fax: 973-256-8341; E-mail: [email protected]; or visit our Website: humanapress.com

Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press, provided that the base fee of US $20.00 per copy is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [0-58829-067-0/04 $20.00].

Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 e-ISBN 1-59259-655-X Library of Congress Cataloging in Publication Data Protein Purification Protocols.--2nd ed. / edited by Paul Cutler. p. ; cm -- (Methods in molecular biology ; v. 244) Includes bibliographical references and index. ISBN 1-58829-067-0 (alk. paper) e-ISBN 1-59259-655-X ISSN 1064-3745 1. Proteins--Purification--Laboratory manuals. [DNLM: 1. Proteins--isolation & purification--Laboratory Manuals. QU 25 P967 2004] I. Cutler, Paul, 1962- II. Methods in molecular biology (Totowa, N.J.) ; v. 244. QP551.P69756 2004 547.7'5046--dc21 2003006803

Cutler_FM_Final 4 10/29/03, 2:43 PM Preface

The first edition of Protein Purification Protocols (1996), edited by Professor Shawn Doonan, rapidly became very successful. Professor Doonan achieved his aims of pro- ducing a list of protocols that were invaluable to newcomers in protein purification and of significant benefit to established practitioners. Each chapter was written by an expe- rienced expert in the field. In the intervening time, a number of advances have war- ranted a second edition. However, in attempting to encompass the recent developments in several areas, the intention has been to expand on the original format, retaining the concepts that made the initial edition so successful. This is reflected in the structure of this second edition. I am indebted to Professor Doonan for his involvement in this new edition and the continuity that this brings. Each chapter that appeared in the original volume has been reviewed and updated to reflect advances and bring the topic into the 21st century. In many cases, this reflects new applications or new matrices available from vendors. Many of these have increased the performance and/or scope of the given method. Several new chapters have been introduced, including chapters on all the currently used protein fractionation and chro- matographic techniques. They introduce the theory and background for each method, providing lists of the equipment and reagents required for their successful execution, as well as a detailed description of how each is performed. The Notes section consti- tutes a reference guide on the issues and pitfalls that may be encountered and provides the means for circumventing or overcoming them effectively. Around the time of the first edition, the concept of proteomics was being forged and has subsequently led to a rapid growth in a new and exciting area of protein isolation and analysis. Techniques such as two-dimensional gel electrophoresis have now entered the mainstream, not only in analysis, but also as a preparative technique for protein characterization. Even newer techniques combine with analytical chromatography as multidimensional separations of proteins and peptides. In combination with mass spec- trometric techniques, these are now the most powerful methods for isolating proteins. Protein Purification Protocols reflects these developments, with chapters encompass- ing all the current thinking. In addition, since the advance of technology means that simple spectrometric detection is no longer the only option for separating proteins, the various methods for detecting proteins are covered. Each chapter is designed to allow a particular step of a purification to be performed in isolation; however, it is understood that a number of steps may need to be run in sequence from initial sample fractionation (e.g., tissue homogenization) to chromatog- raphy and final polishing steps (e.g., buffer exchange). Our book’s format allows for this, and the initial chapter addresses strategies that should place the various method- ologies in context. At the end of the book it was also felt timely to include brief descriptions of how to scale-up purification methods and evaluate the purification of proteins for therapeutic use. These do not rigidly follow the regular pattern for the main body of protocols, but should give an insight into the strategies needed for different final applications. Paul Cutler v

Cutler_FM_Final 5 10/29/03, 2:43 PM Contents

Preface ...... v Contributors ...... xi 1General Strategies Shawn Doonan and Paul Cutler ...... 1 2 Preparation of Extracts From Animal Tissues J. Mark Skehel ...... 15 3 Protein Extraction From Plant Tissues Roger J. Fido, E. N. Clare Mills, Neil M. Rigby, and Peter R. Shewry ...... 21 4 Extraction of Recombinant Protein From Bacteria Anne F. McGettrick and D. Margaret Worrall ...... 29 5 Protein Extraction From Fungi Paul D. Bridge, Tetsuo Kokubun, and Monique S. J. Simmonds...... 37 6 Subcellular Fractionation of Animal Tissues Norma M. Ryan ...... 47 7 Subcellular Fractionation of Plant Tissues: Isolation of Plastids and Mitochondria Alyson K. Tobin and Caroline G. Bowsher ...... 53 8 The Extraction of Enzymes From Plant Tissues Rich in Phenolic Compounds William S. Pierpoint ...... 65 9 Avoidance of Proteolysis in Extracts Robert J. Beynon and Simon Oliver (Revised by Paul Cutler) ...... 75 10 Concentration of Extracts Shawn Doonan ...... 85 11 Making and Changing Buffers Shawn Doonan ...... 91 12 Purification and Concentration by Ultrafiltration Paul Schratter (Revised by Paul Cutler)...... 101 13 Bulk Purification by Fractional Precipitation Shawn Doonan ...... 117 14 Ion-Exchange Chromatography Chris Selkirk ...... 125 15 Hydrophobic Interaction Chromatography Paul A. O’Farrell ...... 133 16 Affinity Chromatography Paul Cutler ...... 139 17 Dye-Ligand Affinity Chromatography Anne F. McGettrick and D. Margaret Worrall ...... 151

vii

Cutler_FM_Final 7 10/29/03, 2:43 PM viii Contents

18 Lectin Affinity Chromatography Iris West and Owen Goldring (Revised by Paul Cutler) ...... 159 19 Immunoaffinity Chromatography Paul Cutler ...... 167 20 Immobilized Metal-Ion Affinity Chromatography Tai-Tung Yip and T. William Hutchens (Revised by Paul Cutler)...... 179 21 Chromatography on Hydroxyapatite Shawn Doonan ...... 191 22 Thiophilic Affinity Chromatography and Related Methods Paul Matejtschuk ...... 195 23 Affinity Precipitation Methods Jane A. Irwin and Keith F. Tipton ...... 205 24 Isoelectric Focusing Reiner Westermeier ...... 225 25 Chromatofocusing Timothy J. Mantle and Patricia Noone ...... 233 26 Size-Exclusion Chromatography Paul Cutler ...... 239 27 Fast Protein Liquid Chromatography David Sheehan and Siobhan O’Sullivan ...... 253 28 Reversed-Phase Chromatography of Proteins William A. Neville ...... 259 29 Extraction of Membrane Proteins Kay Ohlendieck ...... 283 30 Removal of Detergent From Protein Fractions Kay Ohlendieck ...... 295 31 Purification of Membrane Proteins Kay Ohlendieck ...... 301 32 Lyophilization of Proteins Ciarán Ó’Fágáin ...... 309 33 Storage of Pure Proteins Ciarán Ó’Fágáin ...... 323 34 Electroelution of Proteins From Polyacrylamide Gels Michael J. Dunn ...... 339 35 Electroblotting of Proteins From Polyacrylamide Gels Michael J. Dunn ...... 345 36 Two-Dimensional Polyacrylamide Gel Electrophoresis for Proteome Analyses Neil A. Jones...... 353 37 Microscale Solution Isoelectrofocusing: A Sample Prefractionation Method for Comprehensive Proteome Analysis Xun Zuo and David W. Speicher ...... 361

Cutler_FM_Final 8 10/29/03, 2:43 PM Contents ix

38 Practical Column Chromatography Shawn Doonan ...... 377 39 Detection Methods Jacek Mozdzanowski and Sudhir Burman ...... 389 40 Peptide Proteomics Dean E. McNulty and J. Randall Slemmon ...... 411 41 Multidimensional Liquid Chromatography of Proteins Rod Watson and Tim Nadler...... 425 42 Mass Spectrometry David J. Bell ...... 447 43 Purification of Therapeutic Proteins Julian Bonnerjea ...... 455 44 Purification Process Scale-Up Karl Prince and Martin Smith ...... 463 Index ...... 481

Cutler_FM_Final 9 10/29/03, 2:43 PM Contributors

DAVID J. BELL • Genomic and Proteomic Sciences, Medicines Research Center, GlaxoSmithKline Pharmaceuticals, Stevenage, UK ROBERT J. BEYNON • Protein Function Group, Faculty of Veterinary Science, University of Liverpool, Liverpool, UK JULIAN BONNERJEA • Lonza Biologics, Slough, UK CAROLINE G. BOWSHER • School of Biological Sciences, University of Manchester, Manchester, UK PAUL D. BRIDGE • School of Biological and Chemical Sciences, University of London, London, UK and Mycology Section, Royal Botanic Gardens, Kew, Richmond, UK SUDHIR BURMAN • Biopharmaceutical Analytical Development, GlaxoSmithKline Pharmaceuticals, King of Prussia, PA PAUL CUTLER • Genomic and Proteomic Sciences, Medicines Research Center, GlaxoSmithKline Pharmaceuticals, Stevenage, UK SHAWN DOONAN • School of Biosciences, University of East London, UK MICHAEL J. DUNN • Department of Neuroscience, Institute of Psychiatry, King’s College London, London, UK ROGER J. FIDO • Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Bristol, UK OWEN GOLDRING • North East Surrey College of Technology, Ewell, Surrey, IK T. WILLIAM HUTCHENS • LumiCyte Inc., Fremont, CA JANE A. IRWIN • Department of Veterinary Physiology and Biochemistry, University College Dublin, Dublin, Ireland NEIL A. JONES • Genomic and Proteomic Sciences, Medicines Research Center, GlaxoSmithKline Pharmaceuticals, Stevenage, UK TETSUO KOKUBUN • Biological Interactions Section, Royal Botanic Gardens, Kew, Richmond, UK TIMOTHY J. MANTLE • Department of Biochemistry, Trinity College, Dublin, Ireland ANNE F. MCGETTRICK • Department of Biochemistry Trinity College, Dublin, Ireland DEAN E. MCNULTY • Department of Computational, Analytical, and Structural Sciences, GlaxoSmithKline Pharmaceuticals, King of Prussia, PA PAUL MATEJTSCHUK • National Institute for Biological Standards and Control, South Mimms, Potters Bar, UK E. N. CLARE MILLS • Institute of Food Research, Norwich Laboratory, Norwich, UK JACEK MOZDZANOWSKI • Biopharmaceutical Analytical Development, GlaxoSmithKline Pharmaceuticals, King of Prussia, PA TIM NADLER • Applied Biosystems, Framingham, MA WILLIAM A. NEVILLE • Safety Assessment, GlaxoSmithKline Pharmaceuticals, Ware, UK PATRICIA NOONE • Department of Biochemistry, Trinity College, Dublin, Ireland CIARÁN Ó’FÁGÁIN • School of Biotechnology, Dublin City University, Dublin, Ireland PAUL A. O’FARRELL • W. M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

xi

Cutler_FM_Final 11 10/29/03, 2:43 PM xii Contributors

SIMON OLIVER • Department of Biochemistry and Applied Microbiology, UMIST, Manchester, UK SIOBHAN O’SULLIVAN • Department of Biochemistry and Analytical and Biological Chemistry Research Facility, University College Cork, Mardyke, Cork, Ireland KAY OHLENDIECK • Department of Biology, National University of Ireland, Maynooth, Ireland WILLIAM S. PIERPOINT • Rothamsted Research, Harpenden, UK KARL PRINCE • Lonza Biologics, Portsmouth, NH NEIL M. RIGBY • Institute of Food Research, Norwich Laboratory, Norwich, UK NORMA M. RYAN • Department of Biochemistry, University College, Cork, Ireland PAUL SCHRATTER • Millipore Corporation, Bedford, MA CHRIS SELKIRK • Biotherapeutics Development Unit, Cancer Research UK, South Mimms, UK DAVID SHEEHAN • Department of Biochemistry and Analytical and Biological Chemistry Research Facility, University College Cork, Mardyke, Cork, Ireland PETER R. SHEWRY • Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Bristol, UK J. MARK SKEHEL • Genomic and Proteomic Sciences, Medicines Research Center, GlaxoSmithKline Pharmaceuticals, Stevenage, UK MONIQUE S. J. SIMMONDS • Biological Interactions Section, Royal Botanic Gardens, Kew, Richmond, UK J. RANDALL SLEMMON • Department of Genomics and Biotechnology, Pharmacia Corporation, Skokie, IL MARTIN SMITH • Lonza Biologics, Slough, UK DAVID W. SPEICHER • The Wistar Institute, Philadelphia, PA KEITH F. TIPTON • Department of Biochemistry, Trinity College, Dublin, Ireland ALYSON K. TOBIN • Plant Science Laboratory, School of Biology, Sir Harold Mitechell Building, University of St Andrews, St. Andrews, Scotland, UK ROD WATSON • Applied Biosystems, Warrington, UK IRIS WEST • North East Surrey College of technology, Ewell, Surrey, UK REINER WESTERMEIER • Amersham Biosciences Europe GmbH, Freiburg, Germany D. MARGARET WORRALL • Department of Biochemistry and Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland TAI-TUNG YIP • Ciphergen Biosystems Inc., Fremont, CA XUN ZUO • The Wistar Institute, Philadelphia, PA

Cutler_FM_Final 12 10/29/03, 2:43 PM