Methods

in

Molecular Biology

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

For other titles published in this series, go to

www.springer.com/series/7651

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Protein Chromatography
Methods and Protocols

Edited by

Dermot Walls and Sinad T. Loughran

School of Biotechnology and National Centre for Sensor Research,
Dublin City University, Dublin, Ireland

Editors
Dermot Walls, PhD
School of Biotechnology and National Centre
for Sensor Research
Dublin City University
Dublin, Ireland
dermot.walls@dcu.ie

Sinad T. Loughran, PhD

School of Biotechnology and National Centre
for Sensor Research
Dublin City University
Dublin, Ireland
sinead.loughran@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
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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

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Contents

16 Purification of Proteins from Baculovirus-Infected Insect Cells . . . . . . . . . . . . . .

Luke OShaughnessy and Sean Doyle
17 Purification of Poly-Histidine-Tagged Proteins . . . . . . . . . . . . . . . . . . . . . . . . . .
Sinad T. Loughran and Dermot Walls
18 Immobilized Metal Affinity Chromatography/Reversed-Phase Enrichment
of Phosphopeptides and Analysis by CID/ETD Tandem Mass Spectrometry . . . .
Rosana Navajas, Alberto Paradela, and Juan Pablo Albar
19 Tag Removal by Site-Specific Cleavage of Recombinant Fusion Proteins . . . . . . .
Adam Charlton and Michael Zachariou
20 Purification of Antibodies Using Affinity Chromatography . . . . . . . . . . . . . . . . .
Elaine Darcy, Paul Leonard, Jenny Fitzgerald, Martin Danaher,
and Richard O Kennedy
21 Optimized Generation of High-Affinity, High-Specificity
Single-Chain Fv Antibodies from Multiantigen Immunized Chickens . . . . . . . . . .
William J.J. Finlay, Laird Bloom, and Orla Cunningham
22 Measuring ProteinProtein Interactions Using Biacore . . . . . . . . . . . . . . . . . . . .
Paul Leonard, Stephen Hearty, and Richard OKennedy
23 Ultra-Performance Liquid ChromatographyMass Spectrometry of Proteins . . . .
Timothy R. Croley
24 Hydrophobic Interaction Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Philip M. Cummins and Brendan F. OConnor
25 Fast Protein Liquid Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ashkan Madadlou, Siobhan OSullivan, and David Sheehan
26 Shotgun Proteomics: A Qualitative Approach Applying Isoelectric
Focusing on Immobilized pH Gradient and LC-MS/MS . . . . . . . . . . . . . . . . . . .
Laurent Geiser, Ali R. Vaezzadeh, Jacques M.P. Deshusses,
and Denis F. Hochstrasser
27 Shotgun Proteomics: A Relative Quantitative Approach
Using Off-Gel Electrophoresis and LC-MS/MS . . . . . . . . . . . . . . . . . . . . . . . . .
Laurent Geiser, Loc Dayon, Ali R. Vaezzadeh, and Denis F. Hochstrasser
28 Clinical Proteomics: Liquid ChromatographyMass Spectrometry
Purification Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Michael Henry and Paula Meleady
29 Strategies for the Purification of Membrane Proteins . . . . . . . . . . . . . . . . . . . . . .
Sinead Marian Smith
30 A Multi-Step Chromatographic Strategy to Purify Three Fungal
Endo-b-Glucanases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tracey McCarthy and Maria G. Tuohy
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

Mario Lebendiker Protein Expression and Purification Facility, The Wolfson

Centre for Applied Structural Biology, The Hebrew University of Jerusalem,
Jerusalem, Israel
Paul Leonard School of Biotechnology and Biomedical Diagnostics Institute,
Dublin City University, Dublin, Ireland
Sinad T. Loughran School of Biotechnology and National Centre for Sensor
Research, Dublin City University, Dublin, Ireland
Ashkan Madadlou Food Science and Engineering, Tehran University, Karadj, Iran
Tracey McCarthy Department of Biochemistry, School of
Natural Sciences, National University of Ireland, Galway, Ireland
Robert McGrath School of Biotechnology, Dublin City University, Dublin, Ireland
Paula Meleady National Institute for Cellular Biotechnology,
Dublin City University, Dublin, Ireland
John Joseph Milne BioUetikon Ltd, Dublin City University, Dublin, Ireland
Rosana Navajas Laboratorio de Protemica, Centro Nacional de Biotecnologa,
Consejo Superior de Investigaciones Cientficas (CSIC), Madrid, Spain
Brendan F. OConnor National Centre for Sensor Research, and Centre
for Bioanalytical Sciences (CBAS), School of Biotechnology, Dublin City University,
Dublin, Ireland
Ciarn Fgin School of Biotechnology and National Centre for Sensor Research,
Dublin City University, Dublin, Ireland
Richard OKennedy School of Biotechnology and Biomedical Diagnostics Institute,
Dublin City University, Dublin, Ireland
Luke OShaughnessy Department of Biology, National Institute for Cellular
Biotechnology, National University of Ireland Maynooth, Co. Kildare, Ireland
Siobhan OSullivan Department of Biological Sciences,
Cork Institute of Technology, Cork, Ireland; Department of Biochemistry, University
College Cork, Cork, Ireland
Alberto Paradela Laboratorio de Protemica, Centro Nacional de Biotecnologa,
Consejo Superior de Investigaciones Cientficas (CSIC), Madrid, Spain
Barry J. Ryan School of Food Science and Environmental Health, Dublin Institute
of Technology, Dublin, Ireland
David Sheehan Department of Biochemistry, University College Cork, Cork, Ireland
Sinead Marian Smith Institute of Molecular Medicine and Immunology Research
Centre, Trinity College Dublin, Dublin, Ireland
David W. Speicher The Wistar Institute, Philadelphia, PA, USA
Maria G. Tuohy Department of Biochemistry, School of Natural Sciences,
National University of Ireland, Galway, Ireland
Ali R. Vaezzadeh Department of Urology, Childrens Hospital Boston,
Harvard Medical School, Boston, MA, USA
Dermot Walls School of Biotechnology and National Centre for Sensor Research,
Dublin City University, Dublin, Ireland
Michael Zachariou BioMarin Pharmaceutical Inc, Novato, CA, USA