Professional Documents
Culture Documents
in
Molecular Biology
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
wwwwww
Protein Chromatography
Methods and Protocols
Edited by
Editors
Dermot Walls, PhD
School of Biotechnology and National Centre
for Sensor Research
Dublin City University
Dublin, Ireland
dermot.walls@dcu.ie
ISBN 978-1-60761-912-3
e-ISBN 978-1-60761-913-0
DOI 10.1007/978-1-60761-913-0
Springer New York Dordrecht Heidelberg London
Library of Congress Control Number: 2010938718
Springer Science+Business Media, LLC 2011
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.
While the advice and information in this book are believed to be true and accurate at the date of going to press,
neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that
may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein.
Printed on acid-free paper
Humana Press is part of Springer Science+Business Media (www.springer.com)
Preface
Proteins are essential constituents of all organisms, and they participate in virtually every
process within cells. These macromolecules are found in roles that are enzymatic, regulatory, structural, and immunological, to name but a few. In order to elucidate the structure and function of any protein, it is first necessary to purify it, and consequently many
purification schemes and chromatographic methods for the isolation of native proteins
from complex sources have been developed over the years. Every protein has its own
particular sequence of amino acids which is determined by the nucleotide sequence of a
corresponding gene. The last 30 years or so has witnessed revolutionary changes in
experimental biology and specifically in the way that we identify, isolate, and manipulate
individual genes and proteins. Thus, the emergence of recombinant DNA technology,
genomics, and bioinformatics, in particular, means that now theoretically any protein can
be expressed in a tagged and rapidly purifiable recombinant form from a heterologous
host cell.
As in any Methods in Molecular BiologyTM volume, the emphasis here is on the provision
of clear protocol-style chapters that are suitable for newcomers to the field. We first felt that
it was important to include contributions that dealt with generic topics in protein biochemistry, addressing such areas as protein stability and storage, avoiding proteolysis during chromatography, protein quantitation methods including immuno-qPCR, and the contrasting
challenges that microfluidics and scale-up production pose to the investigator.
At a glance, it is clear that more than one third of the chapters concern the generation
and purification of recombinant proteins, reflecting the major contribution of molecular
biology to the field. These largely deal with topics such as recombinant antibody production and the tagging of proteins as a means to enhance their solubility and simplify their
purification on an individual scale or in high-throughput systems. Of course, it is also
expected that a compilation such as this would include the more classical purification
methods that are based on exploiting the physico-chemical properties of the target protein. The reader will therefore find protocol-style chapters on many of the more commonly used methodologies, including proteomic/mass spectrometric techniques. We also
felt that some topics necessitated treatment in an overview-style format due to the need to
encompass a great number of variations that have evolved within these areas (e.g.,
ion-exchange and immunoaffinity chromatography as well as the tagging of recombinant
proteins), often in addition to individual methods-style chapters on those subjects.
However, this compendium of methods in protein chromatography does not pretend to
be comprehensive, and we plead that an attempt to cover the entire potential menu in one
volume would have been futile.
We are indebted to all the authors who have generously given their expertise here.
Our aims were to assemble contributions from experienced scientists who have handson expertise in the field of protein chromatography and to place particular emphasis on
the production of clearly presented step-by-step methodologies, tips, and associated
vi
Preface
explanatory notes. We hope that those who use these methods will succeed in establishing
them in their own laboratories and in troubleshooting any issues that arise. We wish to
extend a particular thanks to the series editor, Prof. John Walker, for his patience, advice,
and encouragement throughout.
Dublin, Ireland
Dermot Walls
Sinad T. Loughran
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
v
ix
Part I Overviews
1 A Digest of Protein Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
Dermot Walls, Robert McGrath, and Sinad T. Loughran
2 Gel-Filtration Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Ciarn Fgin, Philip M. Cummins, and Brendan F. OConnor
3 Immunoaffinity Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Jenny Fitzgerald, Paul Leonard, Elaine Darcy, and Richard OKennedy
4 Avoiding Proteolysis During Protein Chromatography . . . . . . . . . . . . . . . . . . . . 61
Barry J. Ryan
5 Scale-Up of Protein Purification: Downstream Processing Issues . . . . . . . . . . . . . 73
John Joseph Milne
6 Phage Display: A Powerful Technology for the Generation
of High Specificity Affinity Reagents from Alternative Immune Sources . . . . . . . . 87
William J.J. Finlay, Laird Bloom, and Orla Cunningham
7 Engineering Protein Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Ciarn Fgin
8 Microfluidics in Protein Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Frank A. Gomez
9 Tagging Recombinant Proteins to Enhance Solubility and Aid Purification . . . . . 151
Dermot Walls and Sinad T. Loughran
Part II Protocols
10 Storage and Lyophilisation of Pure Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ciarn Fgin
11 Differential Precipitation and Solubilization of Proteins . . . . . . . . . . . . . . . . . . . .
Barry J. Ryan
12 Ion-Exchange Chromatography: Basic Principles and Application
to the Partial Purification of Soluble Mammalian Prolyl Oligopeptidase . . . . . . . .
Philip M. Cummins, Oonagh Dowling, and Brendan F. OConnor
13 Protein Quantitation and Analysis of Purity . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Eva M. Campion, Sinad T. Loughran, and Dermot Walls
14 Purification of Proteins Fused to Glutathione S-Transferase . . . . . . . . . . . . . . . . .
Sandra Harper and David W. Speicher
15 Purification of Proteins Fused to Maltose-Binding Protein . . . . . . . . . . . . . . . . . .
Mario Lebendiker and Tsafi Danieli
vii
179
203
215
229
259
281
viii
Contents
295
311
337
349
369
383
403
419
431
439
449
459
473
485
497
525
Contributors
Juan Pablo Albar Laboratorio de Protemica, Centro Nacional de Biotecnologa,
Consejo Superior de Investigaciones Cientficas (CSIC), Madrid, Spain
Laird Bloom Wyeth Research, Cambridge, MA, USA
Eva M. Campion School of Biotechnology and National Centre for Sensor Research,
Dublin City University, Dublin, Ireland
Adam Charlton Commonwealth Serum Laboratories Ltd, ParkvilleVIC, Australia
Timothy R. Croley US Food and Drug Administration Center for Food Safety and
Applied Nutrition 5100 Paint Branch Parkway, College Park, USA
Philip M. Cummins School of Biotechnology, Dublin City University, Dublin,
Ireland
Orla Cunningham Wyeth Research Ireland, Dublin, Ireland
Martin Danaher Ashtown Food Research Centre, Dublin, Ireland
Tsafi Danieli Protein Expression and Purification Facility, The Wolfson Centre for
Applied Structural Biology, The Hebrew University of Jerusalem, Jerusalem, Israel
Elaine Darcy School of Biotechnology, Dublin City University, Dublin, Ireland
Loc Dayon Biomedical Proteomics Research Group, Department of Structural
Biology and Bioinformatics, Geneva University, Geneva, Switzerland
Jacques M.P. Deshusses Biomedical Proteomics Research Group,
Department of Structural Biology and Bioinformatics, Geneva University,
Geneva, Switzerland
Oonagh Dowling School of Biotechnology, Dublin City University, Dublin, Ireland
Sean Doyle Department of Biology, National Institute for Cellular Biotechnology,
National University of Ireland Maynooth, Co. Kildare, Ireland
William J.J. Finlay Wyeth Research Ireland, Dublin, Ireland
Jenny Fitzgerald School of Biotechnology, Dublin City University, Dublin, Ireland
Laurent Geiser Swiss Centre for Applied Human Toxicology, Geneva University,
Geneva, Switzerland
Frank A. Gomez Department of Chemistry and Biochemistry,
California State University, Los Angeles, CA, USA
Sandra Harper The Wistar Institute, Philadelphia, PA, USA
Stephen Hearty School of Biotechnology and Biomedical Diagnostics Institute,
Dublin City University, Dublin, Ireland
Michael Henry National Institute for Cellular Biotechnology,
Dublin City University, Dublin, Ireland
Denis F. Hochstrasser Swiss Centre for Applied Human Toxicology, and
Departments of Genetics and Laboratory Medicine, Geneva University Hospitals,
School of Pharmaceutical Sciences, Geneva University, Geneva, Switzerland
ix
Contributors