Protein Purification E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Serie: Methods of Biochemical Analysis
• Sprache: Englisch

Seitenzahl: 1535

Veröffentlichungsjahr: 2012

Beschreibung

The authoritative guide on protein purification—now completely updated and revised

Since the Second Edition of Protein Purification was published in 1998, the sequencing of the human genome and other developments in bioscience have dramatically changed the landscape of protein research. This new edition addresses these developments, featuring a wealth of new topics and several chapters rewritten from scratch.

Leading experts in the field cover all major biochemical separation methods for proteins in use today, providing professionals in biochemistry, organic chemistry, and analytical chemistry with quick access to the latest techniques. Entirely new or thoroughly revised content includes:

• High-resolution reversed-phase liquid chromatography

• Electrophoresis in gels

• Conventional isoelectric focusing in gel slabs and capillaries and immobilized pH gradients

• Affinity ligands from chemical and biological combinatorial libraries

• Membrane separations

• Refolding of inclusion body proteins from E. coli

• Purification of PEGylated proteins

• High throughput screening techniques in protein purification

• The history of protein chromatography

Leseprobe

Contents

Cover

Half Title page

Title page

Copyright page

Preface to the Third Edition

Preface to the Second Edition

Preface to the First Edition

Contributors

Part I: Introduction

Chapter 1: Introduction to Protein Purification

Part II: Chromatography

1.1 Introduction

1.2 The Protein Extract

1.3 An Overview of Fractionation Techniques

1.4 Fractionation Strategies

1.5 Monitoring the Fractionation

1.6 The Final Product

1.7 Laboratory Equipment

1.8 References

Chapter 2: Introduction to Chromatography

2.1 Introduction: Basic Concepts and Versions of Chromatography

2.2 The Stationary Phase

2.3 Chromatographic Theory

2.4 Chromatographic Procedures

2.5 Chromatographic Techniques

2.6 On the History of Protein Chromatography

2.7 References

Chapter 3: Gel Filtration: Size Exclusion Chromatography

3.1 Introduction

3.2 Basic Considerations

3.3 Selection of the SEC Support

3.4 Packing the Column

3.5 Sample and Buffer Preparation

3.6 The Chromatographic System

3.7 Applications

3.8 Symbols Used in SEC

3.9 Acknowledgments

3.10 References

Chapter 4: Ion Exchange Chromatography

4.1 Introduction

4.2 The ION Exchange Process

4.3 Charge Properties of Proteins

4.4 The Stationary Phase—the Ion Exchangers

4.5 Nonionic Interactions

4.6 The Mobile Phase: Buffers and Salts

4.7 Experimental Planning and Preparation

4.8 Chromatographic Techniques

4.9 Handling of Isolated Proteins

4.10 Hydroxyapatite Chromatography

4.11 Applications

4.12 Acknowledgments

4.13 References

Chapter 5: High-Resolution Reversed-Phase Chromatography of Proteins

5.1 Introduction

5.2 Fundamentals of RPC

5.3 Development and Optimization of Analytical Separations

5.4 Development and Optimization of Preparative Separations

5.5 Scale-Up

5.6 Column Packing Material

5.7 Column Design

5.8 Practical Recommendations: How to Avoid and Solve Problems

5.9 References

Chapter 6: Hydrophobic Interaction Chromatography

6.1 Introduction

6.2 History

6.3 Nomenclature

6.4 RPC vs HIC

6.5 Theory

6.6 Factors that Impact HIC

6.7 Experimental Design

6.8 Applications

6.9 References

Chapter 7: Immobilized Metal Ion Affinity Chromatography

7.1 Introduction

7.2 Metal Chelate Gels

7.3 Factors Influencing Adsorption and Desorption

7.4 Chromatographic Conditions

7.5 Areas of Use of IMAC

7.6 Applications

7.7 References

Chapter 8: Covalent Chromatography

8.1 Introduction

8.2 Chemical Properties of Thiol Groups

8.3 Thiol-Containing Proteins

8.4 Gels for Covalent Chromatography

8.5 Chromatographic Techniques

8.6 Applications

8.7 References

Chapter 9: Affinity Chromatography

9.1 Introduction

9.2 Affinity Interactions

9.3 Preparation and Evaluation of Affinity Adsorbents

9.4 Immobilization Techniques

9.5 Chromatographic Techniques

9.6 Applications

9.7 References

Chapter 10: Affinity Ligands from Chemical Combinatorial Libraries

10.1 Introduction

10.2 Principles of Ligand Design

10.3 Screening Methodologies

10.4 Summary

10.5 Acknowledgments

10.6 References

Chapter 11: Affinity Ligands from Biological Combinatorial Libraries

11.1 Introduction

11.2 Design of Library

11.3 Selection Methods

11.4 Characterization and Further Engineering of Ligands

11.5 Applications

11.6 References

Part III: Other Separation Methods and Related Techniques

Chapter 12: Membrane Separations

12.1 Introduction

12.2 Membrane Technology

12.3 Static Filtration (SF)

12.4 Tangential Flow Filtration (TFF)

12.5 Virus Filtration

12.6 Membrane Adsorption

12.7 References

Chapter 13: Refolding of Inclusion Body Proteins from E. coli

13.1 Introduction

13.2 Protein Folding in VIVO

13.3 Molecular Simulation of Protein Refolding and Aggregation

13.4 Refolding Methods

13.5 Applications

13.6 References

Chapter 14: Purification of Pegylated Proteins

14.1 Introduction

14.2 General Considerations

14.3 Notes on Selected Separation Methods

14.4 Ion-Exchange Media Specifically Designed for Peg-Proteins

14.5 Summary

14.6 References

Part IV: Electrophoresis

Chapter 15: Electrophoresis in Gels

15.1 Introduction

15.2 Principle

15.3 Gels

15.4 GEL Geometry and Equipment

15.5 Techniques

15.6 Protein Detection Techniques

15.7 References

Chapter 16: Conventional Isoelectric Focusing in Gel Slabs and Capillaries and Immobilized Ph Gradients

16.1 Introduction

16.2 Conventional Isoelectric Focusing in Amphoteric Buffers

16.3 Immobilized pH Gradients

16.4 Capillary Isoelectric Focusing (cIEF)

16.5 Separation of Peptides and Proteins by CZE in Isoelectric Buffers

16.6 Conclusions

16.7 Acknowledgments

16.8 References

Chapter 17: Two-Dimensional Electrophoresis in Proteomics

17.1 Introduction

17.2 Brief Technology Review of 2D Electrophoresis

17.3 Sample Preparation

17.4 Isoelectric Focusing

17.5 SDS Electrophoresis

17.6 Protein Detection

17.7 Image Analysis

17.8 Strategy for Protein Mapping in Proteomics

17.9 Protein Identification and Characterization

17.10 References

Chapter 18: Protein Elution and Blotting Techniques

18.1 Introduction

18.2 Protein Elution

18.3 Protein Blotting

18.4 References

Chapter 19: Capillary Electrophoretic Separations

19.1 Introduction

19.2 Capillary-Type Electrophoretic Instrumentation

19.3 Capillary Electrophoresis of Proteins

19.4 Other Approaches With Applicability to Proteins

19.5 Brief Discussion and Outlook

19.6 Conclusions

19.7 Acknowledgments

19.8 References

Part V: Separation Method Optimization

Chapter 20: High Throughput Screening Techniques in Protein Purification

20.1 Introduction

20.2 Technical Overview

20.3 Microtiter-Based High Throughput Method: Experimental Principle

20.4 Implementation of Design of Experiments in the High Throughput Process Development (HTPD) Area

20.5 Analytics

20.6 Applications

20.7 Summary and Future Trends

20.8 References

Photo Inserts

Index

PROTEIN PURIFICATION

Copyright © 2011 by John Wiley & Sons, Inc. All rights reserved

Published by John Wiley & Sons, Inc., Hoboken, New JerseyPublished simultaneously in Canada

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Library of Congress Cataloging-in-Publication Data:

Protein purification : principles, high resolution methods, and applications/edited by Jan-Christer Janson. — 3rd ed. p. cm. Includes index. ISBN 978–0–471–74661–4 (cloth) 1. Proteins-Purification. 2. Chromatographic analysis. 3. Electrophoresis. I. Janson, Jan-Christer.QP551.P69754 2011 572’.6—dc22 2010033316

PREFACE TO THE THIRD EDITION

Most will agree that the major achievement in bioscience since 1998, when the second edition of this book was published, is sequencing of the human genome. Rather than diminishing interest in proteins, this has led to a revival in protein exploration and an intensive search for better understanding of molecular processes in health and disease. During this time, industrial exploitation of proteins in healthcare has hardly declined. The application of monoclonal antibodies targeted against rheumatoid arthritis and cancer has been booming, many second- and third-generation bio-pharmaceuticals have been approved, and modern technologies for vaccine production based on protein engineering and cell culture are being developed on a wide front.

There are approximately 21,000 protein-encoding genes, and the human proteome is much larger than this. Although mapping the genome revealed what was in the box, the jigsaw puzzle is far from complete. Several major research projects exemplify the revitalized interest in proteins. One is the Protein Atlas initiative (www.protematlas.org), aimed at providing a comprehensive database of high resolution microscopic images identifying proteins in normal and cancer tissues. Others involve an ever-widening range of refined tools exploiting protein profiling micro arrays, surface plasmon resonance, mass spectrometry, ELISA, quantitative 2D electrophoresis, and so on. Many technologies are aimed at parallel processing of thousands of targets, and this is profoundly changing the way structural biology projects are managed. Streamlined, miniaturized, automated high throughput (HTP) protocols are becoming the standard, but there is still a fundamental need for protein expression and purification, not least for X-ray structural studies. Many “proteomic” projects exploit high throughput purification of tagged proteins or antibodies.

On the industrial side, particular in healthcare, protein production is rapidly maturing. Platform technologies are being applied both upstream and downstream, allowing faster and leaner implementation as well as better control. Expression of monoclonal antibodies in mammalian cells is at the multi-gram per liter level, with cell densities of more than twenty million per milliliter, specific productivity over 20 picograms per cell per day, in bioreactors with capacities up to 20,000 liters. This several-hundred-fold increase in productivity has changed the pressures on downstream purification, resulting in the development of very high capacity chromatography media for product capture and highly selective media (frequently “multimodal”) for polishing. Downstream purification of biopharmaceuticals uses platform modules for assuring virus safety and for removal of host cell proteins, aggregates and critical contaminants. Regulatory agencies are encouraging greater understanding and control of production processes, a quality by design (QbD) doctrine, and the use of modern risk management techniques and experimental design—all of which is impacting the development of purification methods.

Compared to the second edition of this book, four chapters have been deleted (Chromatofocusing, Affinity Partitioning, Immunoelectrophoresis, and Large-Scale Electrophoresis). Three chapters have been totally rewritten by new authors: Chapter 5 (High Resolution Reversed-Phase Liquid Chromatography of Proteins), Chapter 15 (Electrophoresis in Gels), Chapter 16 (Conventional Isoelectric Focusing in Gel Slabs and Capillaries and Immobilized pH Gradients). Six new chapters have been added: Chapter 10 (Affinity Ligands from Chemical Combinatorial Libraries), Chapter 11 (Affinity Ligands from Biological Combinatorial Libraries), Chapter 12 (Membrane Separations), Chapter 13 (Refolding of Inclusion Body Proteins from E. coli), Chapter 14 (Purification of PEGylated Proteins), and Chapter 20 (High Throughput Screening Techniques in Protein Purification). These new chapters have been written by leading experts in their respective fields. All other chapters have been thoroughly revised and updated regarding recent applications. A new section on the history of protein chromatography has been added to Chapter 2 (Introduction to Chromatography).

It is my hope that the third edition will receive the same overwhelmingly positive response as the first and second editions, and I would like to express my appreciation to all contributing authors and to Ms Anita Lekhwani and her staff at John Wiley & Sons, Inc., Hoboken, New Jersey, for their patience and never-failing support of this project.

JAN-CHRISTER JANSON

PREFACE TO THE SECOND EDITION

Since 1989, when the first edition of this book was launched, the development of biosciences has meant a revival of protein chemistry in the wake of the molecular biology revolution and the HUGO project. The total genome of baker’s yeast is now sequenced, that of E. coli is not far behind, and within a not too distant future the feat of the total mapping of the human genome, which at the beginning seemed fictitious, is now within reach. This means that the attention of the world’s bioscientific community will again, as in the 1960s and most of the 1970s, focus on the structure and function of the proteins. The PROTEOME era has thus begun, and with it follows the need of more efficient and more selective tools for the separation, isolation, and purification of the gene products, the proteins.

The development of new chromatographic separation media since 1989 has mainly been focused toward improvements demanded primarily by process development engineers in the biopharmaceutical industry. This has resulted in media with higher efficiencies, leading to shorter cycle times, primarily based on suspension polymerized styrene-divinylbenzene polymers with optimized internal pore size distributions, some allowing partial convective flow through the particles. This trend has received its ultimate solution in totally perfusive systems based on stacked membranes, or continuous “monolithic” columns made of cross-linked polymers, derivatized with various kinds of protein adsorptive groups. New composite media have been introduced primarily to increase the industrial applicability of size exclusion chromatography of proteins but also to increase binding capacity in, for example, ion exchange chromatography. The concept of “solid diffusion” in highly ionic group substituted composite media is still awaiting its physicochemical explanation.

The demand for systems allowing direct capture of target proteins directly from whole cultures or cell homogenates, resulting in fewer process steps and concomitantly higher yields, has led to a revival of the fluidized bed concept. However, now optimized with regard to the design of both media and columns by the introduction of the more efficient one cycle technique called expanded bed adsorption.

As long as scientists have been engaged in the isolation and purification of proteins from crude extracts, there has been a demand for media with higher adsorptive selectivities. The extremely high variability in protein surface structure as well as their wide range of functional stabilities, makes it necessary for every protein chemist to have a stock of several different separation media, ion exchangers, hydrophobic interaction media, and a variety of general affinity media. Literature survey data presented in some of the chapters of this book reveal that on average somewhere between three and four steps are required to purify a protein to homogeneity. The hope for one-step purifications raised by the introduction of immobilized monoclonal antibodies has not yet been fulfilled. However, there is a renewed opportunity at hand to increase the selectivity of immobilized ligands in affinity chromatography and thus decrease the number of steps in the purification process. This opportunity has been raised by the recent rapid development in the design of a large variety of chemical and biological combinatorial libraries and high-speed screening technologies. It is easy to predict that over the next few years there will be an unprecedented number of new highly selective ligands, monospecific as well as group specific, introduced for the synthesis of new protein separation media.

Compared to the first edition of this book, there exists one additional chapter (Chapter 18) on large-scale electrophoretic processes. Three chapters (Chapters 15, 16, and 17) have been totally rewritten. Chapters 15 and 16 by new authors. Most other chapters have been thoroughly revised, and all have been updated regarding recent applications.

It is our hope that this new edition will receive the same overwhelmingly positive response as the first edition, and we would like to express our appreciation to Dr. Edmund H. Immergut and the staff of VCH Publishers, now John Wiley & Sons, Inc., for their patience and never-failing support of this project.

JAN-CHRISTER JANSONLARS RYDÉN

PREFACE TO THE FIRST EDITION

Over the last two decades the scientific community has witnessed an unprecedented expansion within the biosciences and biotechnology. This expansion has been to a large extent driven by advances in several key areas, most notably recombinant DNA technology, hybridoma and cell culture techniques and, finally, in biochemical separation methods. This book is a description of the current status of one of these areas: modern techniques for protein purification and analysis.

The research on which the progress in separation techniques is based has been conducted both in university departments, devoted to basic research, and in industrial laboratories whose main concern is the development of new equipment and tools. In many cases the two communities have cooperated to their mutual benefit. In fact, a great number of the products now available for the separation and purification of proteins, such as chromatographic media with a wide range of selectivities and efficiencies, as well as equipment for electrophoretic separation and analysis, were originally developed in a university setting. This book is also the result of a joint effort between university researchers, in particular at Uppsala University, and the research staff of a company, Pharmacia LKB Biotechnology. Although it is thus a product of this condition of mutual benefit, the ambition has not been to give a selective description of methods or materials from a single commerical source, but rather to give an unbiased account of all key techniques in the field.

Today it is to a great extent possible to base the separation of proteins on knowledge of their molecular properties, structural as well as functional. Suggestions on how to solve a separation problem can best be made if data on protein structure and function, including particular structural details, is available. Conversely, results from the application of a particular separation method can often be interpreted in terms of molecular properties of the protein under study. Throughout the text of this book, separation results are related to protein properties, often in a detailed manner. We are the first generation to be on the verge of rational protein management.

Starting with this general concept, we have aimed at providing students, teachers and research workers in biomedicine, bioscience and biotechnology with a concise and practical treatise covering, in a single volume, all important chromatographic and electrophoretic techniques used in preparative and analytical protein chemistry. The book contains a general introductory chapter on protein preparative work, Chapter 1, where the key concepts are introduced. Similarly, a general introduction to chromatography is given in Chapter 2 and an introduction to analytical electrophoresis in Chapter 12. The major chromatographic and electrophoretic techniques are presented in individual chapters, including one chapter on affinity partitioning in aqueous polymer two-phase systems.

No single person can today be even close to acquiring the amount of experience necessary to describe with confidence the wealth of techniques and methods which makes up the arsenal for protein separations. We have thus chosen to produce a multi-author volume recruiting expertise from the entire field. All chapters have, however, been thoroughly worked through by the editors to achieve a reasonable uniformity of style and organization. Each chapter deals first with the theory and underlying principles of each separation technique, followed by a section on methodology, and ends with a number of representative application examples described in detail.

The preparation of this book has been a matter of several years. We would like to thank the authors for their cooperation, from the first planning stage to the last phase of updating and addition. We would also like to thank our editors at VCH Publishers in New York, in particular Dr. Edmund H. Immergut who took the first initiative and who followed the project up to its realization. The management and staff of Pharmacia LKB Biotechnology are thanked for their cooperation and support which allowed the selling price to be considerably reduced. Many staff members have made invaluable contributions to the final result, which are gratefully acknowledged. We also thank Elizabeth Hill and Ursula Snow for their contributions in the early phase of the project; Inger Galvér, Gull-Maj Hedén, Inga Johansson and Madeleine de Sharengrad for secretarial work; Bengt Westerlund for handling the computer programmes for the chemical structures; Uno Skatt and Lilian Forsberg for producing a number of the illustrations; and David Eaker and John Brewer for keeping our freedom with the English language within limits. Finally, we would like to add that we are well aware that much of our own efforts, occasional achievements and sometimes hardwon experience, as well as that of several of the other authors of this book, spring from the tree planted long ago by The Svedberg and Arne Tiselius, and later kept alive by Jerker Porath and Stellan Hjertén and many of their colleagues and pupils through fifty years of separation science at Uppsala University. We offer this book as the latest fruit of this tree, hopefully to be enjoyed by many.

JAN-CHRISTER JANSON LARS RYDÉNUppsala, Sweden, June 21, 1989

CONTRIBUTORS

Francisco Batista-Viera, Cátedra de Bioquímica, Dpto. de Biociencias, Facultad de Quimica Gral. Flores 2124. Casilla de Correo 1157, Montevideo, Uruguay

Herbert Baumann, GE Healthcare Bio-Sciences AB, SE-751 84 Uppsala, Sweden

Makonnen Belew, GE Healthcare Bio-Sciences AB, SE-751 84 Uppsala, Sweden

Eggert Brekkan, GE Healthcare Bio-Sciences AB, SE-751 84 Uppsala, Sweden

Jan Carlsson, Department of Physical & Analytical Chemistry, Uppsala University, Box 579, SE-751 23 Uppsala, Sweden

Enrique Carredano, GE Healthcare Bio-Sciences AB, SE-751 84 Uppsala, Sweden

Kjell-Ove Eriksson, GE Healthcare Bio-Sciences AB, SE-751 84 Uppsala, Sweden

Bo Ersson, Medicago AB, Danmark-Berga 13, SE-755 98 Uppsala, Sweden

Elisa Fasoli, Department of Chemistry, Materials and Chemical Engineering, “Giulio Natta,” Politecnico di Milano, Via Mancinelli 7, 20131 Milano, Italy

Conan J. Fee, Biomolecular Interaction Centre and Department of Chemical and Process Engineering, University of Canterbury, Private Bag 4800, Christchurch 8020, New Zealand

Angelika Görg, Department of Proteomics, Technische Universität München, D-85350 Freising-Weihenstephan, Germany

Uwe Gottschalk, Sartorius Stedim Biotech GmbH, August-Spindler-Straße 11, D-37079 Göttingen, Germany

Lars Hagel, GE Healthcare Bio-Sciences AB, SE-751 84 Uppsala, Sweden

Irwin Hirsh, Novo Nordisk AS, Nybrovej 80, 2820 Gentofte, Denmark

Jan-Christer Janson, Department of Physical and Analytical Chemistry, Uppsala University, Box 579, S-751 23 Uppsala, Sweden

Zuwei Jin, GE Healthcare Life Sciences, Building 1, No 1 Huatuo Road, Zhangjiang Hi-Tech Park, Pudong New Area, Shanghai 201203, China

Maik W. Jornitz, Sartorius Stedim North America Inc., 5 Orville Drive, Bohemia, New York 11716, USA

Jan Åke Jönsson, Center for Analysis and Synthesis, Department of Chemistry, Lund University, Box 124, S-22100 Lund, Sweden

Lennart Kågedal, GE Healthcare Bio-Sciences AB, SE-751 82 Uppsala, Sweden

Evert Karlsson, Department of Biochemistry and Organic Chemistry, Uppsala University, Box 576, SE-751 23 Uppsala, Sweden

Karol M. Lacki, GE Healthcare Bio-Sciences AB, SE-751 84 Uppsala, Sweden

Zheng Liu, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China

Diannan Lu, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China

Per-Åke Nygren, Division of Molecular Biotechnology, School of Biotechnology, Royal Institute of Technology (KTH), SE-106 91 Stockholm, Sweden

Sylvia Winkel Pettersson, Eka Chemicals AB/Akzo Nobel, Bohus, Sweden

Pier Giorgio Righetti, Department of Chemistry, Materials and Chemical Engineering, “Giulio Natta,” Politecnico di Milano, Via Mancinelli 7, 20131 Milano, Italy

Sabina Carla Righetti, Department of Chemistry, Materials and Chemical Engineering, “Giulio Natta,” Politecnico di Milano, Via Mancinelli 7, 20131 Milano, Italy

Lars Rydén, Centre for Sustainable Development (CSD) Uppsala, Uppsala University, Villavägen 16, SE-752 36 Uppsala, Sweden

Zhiguo Su, State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080, China

Wolfgang Thormann, Department of Clinical Pharmacology, University of Bern, Murtenstraße 35, CH-3010 Bern, Switzerland

James M. Van Alstine, GE Healthcare Bio-Sciences AB, 751 84 Uppsala, Sweden

Joachim K. Walter, InnoBiologics Sdn Bhd, Lot 1, Persiaran Negeri BBN, 71800 Nilai, Malaysia

Reiner Westermeier, SERVA Electrophoresis GmbH, Carl-Benz-Strasse 7, D-69115 Heidelberg, Germany

PART I

INTRODUCTION

CHAPTER 1

INTRODUCTION TO PROTEIN PURIFICATION

BO ERSSON

Medicago AB, Danmark-Berga 13, SE-755 98 Uppsala, Sweden

LARS RYDÉN

Centre for Sustainable Development (CSD) Uppsala, Uppsala University, Villavägen 16, SE-752 36 Uppsala, Sweden

JAN-CHRISTER JANSON

Department of Physical and Analytical Chemistry, Uppsala University, Box 579, S-751 23 Uppsala, Sweden

1.1 Introduction

1.2 The Protein Extract

1.2.1 Choice of Raw Material

1.2.2 Extraction Methods

1.2.3 Extraction Medium

1.2.3.1 pH

1.2.3.2 Buffer Salts

1.2.3.3 Detergents and Chaotropic Agents

1.2.3.4 Reducing Agents

1.2.3.5 Chelators or Metal Ions

1.2.3.6 Proteolytic Inhibitors

1.2.3.7 Bacteriostatics

1.3 An Overview of Fractionation Techniques

1.3.1 Precipitation

1.3.2 Electrophoresis

1.3.3 Chromatography

1.3.4 Expanded Bed Adsorption

1.3.5 Membrane Adsorption

1.4 Fractionation Strategies

1.4.1 Introductory Comments

1.4.2 Initial Fractionation

1.4.2.1 Clarification by Centrifugation and/or Microfiltration

1.4.2.2 Ultrafiltration

1.4.2.3 Precipitation

1.4.2.4 Liquid-Liquid Phase Extraction

1.4.3 The Chromatographic Steps

1.4.3.1 Choice of Adsorbent

1.4.3.2 The Order of the Chromatographic Steps

1.4.4 The Final Step

1.5 Monitoring the Fractionation

1.5.1 Assay of Biological Activity

1.5.2 Determination of Protein Content

1.5.3 Analytical Gel Electrophoresis

1.6 The Final Product

1.6.1 Buffer Exchange

1.6.2 Concentration

1.6.3 Drying

1.7 Laboratory Equipment

1.7.1 General Equipment

1.7.2 Equipment for Homogenization

1.7.3 Equipment for Chromatography

1.7.3.1 Column Design

1.7.3.2 Pumps and Fraction Collectors

1.7.3.3 Monitoring Equipment

1.7.3.4 Chromatography Systems

1.7.4 Equipment for Chromatographic and Electrophoretic Analyses

1.8 References

1.1 INTRODUCTION

The development of techniques and methods for the separation and purification of biological macromolecules such as proteins has been an important prerequisite for many of the advancements made in bioscience and biotechnology over the past five decades. Improvements in materials, utilization of computerized instruments, and an increased use of in vivo tagging have made protein separations more predictable and controllable, although many still consider purification of non-tagged proteins more an art than a science. However, gone are the days when an investigator had to spend months in search of an efficient route to purify an enzyme or hormone from a cell extract. This is a consequence of the development of new generations of chromatographic media with increased efficiency and selectivity as well as of new automated chromatographic systems supplied with sophisticated interactive software packages and data bases. New electrophoresis techniques and systems for fast analysis of protein composition and purity have also contributed to increasing the efficiency of the evaluation phase of the purification process.

In the field of chromatography, the development of new porous resin supports, new crosslinked beaded agaroses, and new bonded porous silicas has enabled rapid growth in high resolution techniques (high performance liquid chromatography, HPLC; fast protein liquid chromatography, FPLC), both on an analytical and laboratory preparative scale as well as for industrial chromatography in columns with bed volumes of several hundred liters. Expanded bed adsorption enables rapid isolation of target proteins, directly from whole cell cultures or cell homogenates. Another field of increasing importance is micropreparative chromatography, a consequence of modern methods for amino acid and sequence analysis requiring submicrogram samples. The data obtained are efficiently exploited by recombinant DNA technology, and biological activities previously not amenable to proper biochemical study can now be ascribed to identifiable proteins and peptides.

A wide variety of chromatographic column packing materials such as gel-filtration media, ion exchangers, reversed phase packings, hydrophobic interaction adsorbents, and affinity chromatography adsorbents are today commercially available. These are identified as large diameter media (90–100 μm), medium diameter media (30–50 μm) and small diameter media (5–10 μm) in order to satisfy the different requirements of efficiency, capacity, and cost.

However, not all problems in protein purification are solved by the acquisition of sophisticated laboratory equipment and column packings that give high selectivity and efficiency. Difficulties still remain in finding optimum conditions for protein extraction and sample pretreatment, as well as in choosing suitable methods for monitoring protein concentration and biological activity. These problems will be discussed in this introductory chapter. There will also be an overview of different protein separation techniques and their principles of operation. In subsequent chapters, each individual technique will be discussed in more detail. Finally, some basic equipment necessary for efficient protein purification work will be described in this chapter.

Several useful books covering protein separation and purification from different points of view are available on the market or in libraries (1–3). In “Methods of Enzymology,” for example, in older volumes 22, 34, 104, and 182 (4–7), but particularly in the most recent volume, 463 (8), a number of very useful reviews and detailed application reports will be found. The booklets available from manufacturers regarding their separation equipment and media can also be helpful by providing detailed information regarding their products.

1.2 THE PROTEIN EXTRACT

1.2.1 Choice of Raw Material

In most cases, interest is focused on one particular biological activity, such as that of an enzyme, and the origin of this activity is often of little importance. Great care should therefore be taken in the selection of a suitable source. Among different sources there might be considerable variation with respect to the concentration of the enzyme, the availability and cost of the raw material, the stability of the enzyme, the presence of interfering activities and proteins, and difficulties in handling a particular raw material. Very often it is compelling to choose a particular source because it has been described previously in the literature. However, sometimes it is advantageous to consider an alternative choice.

Traditional animal or microbial sources have today, to a large degree, been replaced by genetically engineered microorganisms or cultured eukaryotic cells. Protein products of eukaryotic orgin, cloned and expressed in bacteria such as Escherichia coli, may either be located in the cytoplasm or secreted through the cell membrane. In the latter case they are either collected inside the periplasmic space or they are truly extracellular, secreted to the culture medium. Proteins that accumulate inside the periplasmic space may be selectively released either into the growth medium by changing the growth conditions (9), or following cell harvesting and washing of the resuspended cell paste. At this stage, a considerable degree of purification has already been achieved by choosing a secreting strain as illustrated in Figure 1.1. In connection with the cloning, the recombinant protein may be equipped with an “affinity handle” such as a His-tag or a fusion protein such as Protein A, glutathione-S-transferase, or maltose binding protein in order to facilitate purification. The handle is often designed such that it can be cleaved off using highly specific proteolytic enzymes. Proteins of eukaryotic origin, and some virus surface proteins are often glycosylated why eukaryotic host cells have to be chosen for their production.

Figure 1.1 Location and approximate numbers of proteins in E. coli.

1.2.2 Extraction Methods

Some biological materials themselves constitute a clear or nearly clear protein solution suitable for direct application to chromatography columns after centrifugation or filtration. Examples include blood serum, urine, milk, snake venoms, and—perhaps most importantly—the extracellular medium after cultivation of microorganisms and mammalian cells, as mentioned above. It is normally an advantage to choose such a starting material because of the limited number of components and also because extracellular proteins are comparatively stable. Some samples, such as urine or cell culture supernatants, are normally concentrated before purification begins.

In most cases, however, it is necessary to extract the activity from a tissue or a cell paste. This means that a considerable number of contaminating molecular species are set free, and proteolytic activity will make the preparation work more difficult. The extraction of a particular protein from a solid source often involves a compromise between recovery and purity. Optimization of extraction conditions should favor the release of the desired protein and leave difficult-to-remove contaminants behind. Of particular concern is to find conditions under which the already extracted protein is not degraded or denatured while more is being released.

Various methods are available for the homogenization of cells or tissues. For further details and discussions the reader is referred to the paper by Kula and Schütte (10). The extraction conditions are optimized by systematic variation of parameters such as the composition of the extraction medium (see below), time, temperature, and type of equipment used.

The proper design of an extraction method thus requires preliminary experiments in which aliquots are taken at various time intervals and analyzed for activity and protein content. The number of parameters can be very large, so this part of the work has to be kept within limits by applying proper judgment. However, it is not recommended to accept a single successful experiment. Further investigations of the required extraction time, in particular, often pays in the long run. The number of optimization experiments can be reduced considerably by using chemometrics (multivariate analysis), for which there are computer programs available (www.chemometrics.com/software).

The major problems confronted when preparing a protein are in general denaturation, proteolysis, and contamination with pyrogens, nucleic acids, bacteria, and viruses. These can be limited by appropriate choice of the extraction medium, as we shall show. However, we can already state that many of the above problems can be reduced by short preparation times and low temperatures. It is therefore good biochemical practice to carry out the first preparation steps as fast as possible and at the lowest possible temperature. However, low temperatures are not always necessary and are sometimes inconvenient. The working temperature is therefore one of the parameters that should be optimized carefully, especially if a preparation is to be done routinely in the laboratory or if it is going to be scaled up to pilot or production scale.

The extract must be clarified by centrifugation and/or by filtration before submission to column chromatography. A preparative laboratory centrifuge is normally sufficient for this step.

A common phenomenon when working with intracellularly expressed recombinant proteins is their tendency to accumulate as insoluble aggregates known as inclusion bodies, which have to be solubilized and refolded to recover their native state. At first glance, the formation of insoluble aggregates in the cytoplasm might be considered a major problem. However, as the inclusion bodies seem to be fairly well defined with regard to both particle size and density (11), they should provide a unique means for rapid and efficient enrichment of the desired protein simply by low speed fractional centrifugation and washing of the resuspended sediment. The critical step is solubilization and refolding, often combined with chromatographic purification under denaturating conditions in the presence of high concentrations of urea or guanidine hydrochloride. This area is treated in more detail in Chapter 13 and has recently been reviewed by Burgess (12).

1.2.3 Extraction Medium

To arrive at a suitable composition for the extraction medium it is necessary initially to study the conditions at which the protein of interest is stable and secondly, where it is most efficiently released from the cells or tissue. The final choice is usually a compromise between maximum recovery and maximum purity. The following factors have to be taken into consideration: pH, buffer salts, detergents/chaotropic agents, reducing agents, chelators or metal ions, proteolytic inhibitors, and bacteriostatics.

1.2.3.1 pH Normally, the pH value is chosen such that the activity of the protein is at a maximum. However, it should be noted that this is not always the pH that gives the most efficient extraction, nor is it necessarily the pH of maximum stability. For example, trypsin has an activity optimum at pH 8–9, but is much more stable at pH 3, where autolysis is avoided. The use of extreme pH values, for example, for the extraction of yeast enzymes in 0.5 M ammonia, is some times very efficient and is acceptable for some proteins with out causing too much denaturation.

1.2.3.2 Buffer Salts Most proteins are maximally soluble at moderate ionic strengths, 0.05–0.1, and these values are chosen if the buffer capacity is sufficient. Suitable buffer salts are given in Table 1.1.

TABLE 1.1Buffer Salts Used in Protein Work

Buffer pK values Properties

Sodium acetate4.75Sodium bicarbonate6.50, 10.25Sodium citrate3.09, 4.75, 5.41Binds Ca2+Ammonium acetate4.75, 9.25VolatileAmmonium bicarbonate6.50, 9.25, 10.25VolatileTris-hydrochloride8.21Sodium phosphate1.5,7.5, 12.0Tris-phosphate7.5, 8.21

Buffer concentration refers to total concentration of buffering species. Buffer pH should be as close as possible to the pKa value, and not more than one pH unit from the pKa.

An acceptable buffer capacity is obtained within one pH unit from the pKa values given. The proteins as such also act as buffers, and the pH should be checked after addition of large amounts of proteins to a weakly buffered solution. Some extractions do not give rise to acids and bases and thus do not need a high buffer capacity. In other cases this might be necessary, and occasional control of the pH value of an extract is recommended.

1.2.3.3 Detergents and Chaotropic Agents In many extractions the desired protein is bound to membranes or particles, or is aggregated due to its hydrophobic character. In these cases one should reduce the hydrophobic interactions by using either detergents or chaotropic agents (not both!). Some of the commonly used detergents are listed in Table 1.2. Several of them do not denature globular proteins or interfere with their biological activity. Others, such as sodium dodecyl sulfate (SDS), will do that. Quite often it is not necessary to continue using a detergent in the buffer after the first step(s) in the purification, so its use is restricted to the extraction medium. In other cases it might be necessary to use a detergent throughout the whole preparation process, leading to the final purification of a protein-detergent complex. More information about detergents, including their chemical structures, can be found in Reference 13.

TABLE 1.2Detergents Used for Solubilization of Proteins

Detergents are amphipathic molecules. When their concentration increases they will eventually aggregate; that is, they will form micelles at the so-called critical micelle concentration (CMC). Because micelles often complicate purification procedures, in particular column chromatography, detergent concentrations below the CMC should be used.

Instead of using detergents to dissolve aggregates, chaotropic agents such as urea or guanidine hydrochloride, or moderately hydrophobic organic compounds such as ethylene glycol, can be tried. Urea and guanidine hydrochloride have proven particularly useful for the extraction and solubilization of inclusion bodies (12).

1.2.3.4 Reducing Agents The redox potential of the cytosol is lower than that of the surrounding medium where atmospheric oxygen is present. Intracellular proteins often have exposed thiol groups and these might become oxidized in the purification process. Thiol groups can be protected by reducing agents such as 1,4-dithioerythritol (DTE), dithiothreitol (DTT) or mercaptoethanol (Table 1.3). Normally, 10–25 mM concentrations are sufficient to protect thiols without reducing internal disulfides. In other cases a higher concentration might be needed (14). Ascorbic acid is some times added to polyphenol containing crude plant extracts in order to avoid oxidation and miscoloration.

TABLE 1.3Reducing Agents

AgentStructureMercaptoethanolHS—CH2—CH2—OH1,4–Dithioerythritol (DTE)1,4–Dithiothreitol (DTT)Ascorbic acid

1.2.3.5 Chelators or Metal Ions The presence of heavy metal ions can be detrimental to a biologically active protein, for two main reasons. They can enhance the oxidation of thiols by molecular oxygen and can form complexes with specific groups, which may cause problems. Heavy metals can be trapped by chelating agents. The most commonly used is ethylenediamine tetraacetic acid (EDTA) in the concentration range 10–25 mM. An alternative is ethylene glycol tetraacetic acid (EGTA), which is more specific for calcium. It should be noted that EDTA is a buffer. It is therefore best to add EDTA before final pH adjustment. The chelating capacity of EDTA increases with increasing pH.

In other cases, stabilizing metal ions are needed. Many proteins are stabilized by calcium ions. However, the divalent ions calcium and magnesium are trapped by EDTA and cannot be used in combination with this chelator.

1.2.3.6 Proteolytic Inhibitors The most serious threats to protein stability are the omnipresent proteases. The simplest safeguard against proteolytic degradation is normally to work quickly at low temperatures. An alternative, or added, precaution is to make use of protease inhibitors (Table 1.4), especially in connection with the extraction step. Often there is a need for a combination of inhibitors, for example, for both serine proteases and metalloproteases. In general, protein inhibitors are expensive, which may limit their use in large-scale applications. Proteolysis can also be reduced by rapid extraction of the fresh homogenate in an aqueous polymer two-phase system (15) or by adsorption of the proteases to hydrophobic interaction adsorbents (16). Sometimes it is sufficient to adjust the pH to a value at which the proteases are inactive, but where the stability of the protein to be purified is maintained. A classical example is the purification of insulin from the pancreas.

TABLE 1.4Proteolytic Inhibitors

InhibitorEnzymes InhibitedWorking ConcentrationDiisopropyl fluorophosphate (DFP)Serine proteases(avoid DFP)Phenylmethylsulfonyl fluoride (PMSF)Serine proteases0.5–1 mMEthylenediamine tetraacetate (EDTA)Metal-activated proteases~5 mMCysteine reagentsCysteine-dependent proteases(varying)Pepstatin AAcid proteases1 μMLeupeptinSerin proteases1 μM

1.2.3.7 Bacteriostatics It is wise to take precautions to avoid bacterial growth in protein solutions. The simplest remedy here is to use sterile filtered buffer solutions as routine in the laboratory. This will also reduce the risk of bacterial growth in columns. A common practice for avoiding bacterial growth in chromatographic columns is to allow the column to flow at a reduced rate, even when it is not in operation. Some buffers are more likely than others to support bacterial growth, such as phosphate, acetate, and carbonate buffers at neutral pH values. Buffers at pH 3 and below or at pH 9 and above usually prevent bacterial growth, but may occasionally allow growth of molds.

Whenever possible it is recommended to add an antimicrobial agent to the buffer solutions. Often used are sodium azide at 0.001 M or merthiolate at 0.005%, or alcohols such as n-butanol at 1%. Sodium azide has the drawback that it is a nucleophilic substance and binds metals. In cases where these substances may interfere with activity measurements or the chromatography itself, it is always possible to add the substances to solutions of the protein to be stored.

1.3 AN OVERVIEW OF FRACTIONATION TECHNIQUES

In early work, complex protein mixtures were fractionated mainly by adsorption and precipitation methods. These methods are still used today as preliminary steps for initial fractionation or for concentration of sample solutions. Preparative electrophoretic and chromatographic techniques developed during the 1950s and 1960s made possible rational purification protocols and laid the foundation for the situation we have today. The following sections give a short overview of the various techniques normally used in preparative biochemical work. Chapter 2 contains an introduction to chromatography, including a historical review, and Chapter 15 gives an introduction to electrophoresis. Each individual chromatographic and electrophoretic separation technique is then treated in detail in subsequent chapters.

1.3.1 Precipitation

Precipitation of a protein in an extract may be achieved by adding salts, organic solvents, or organic polymers, or by varying the pH or temperature of the solution. The most commonly used precipitation agents are listed in Table 1.5. The strength of a particular ion as a precipitation agent is shown by its position in the so-called Hofmeister series:

TABLE 1.5Precipitation Agents

AgentTypePropertiesAmmonium sulfateSaltEasily soluble, stabilizingSodium sulfateSaltEthanolSolventFlammable, risk of denaturationAcetoneSolventFlammable, risk of denaturationPolyethylene glycol (PEG)PolymerUncharged, unflammable

The so-called antichaotropic ions to the left are the most efficient salting out agents. They are efficient water molecule binders, thus increasing the hydrophobic effect in the solution and promoting protein aggregation by facilitating the association of hydrophobic surfaces. The chaotropic salts on the right-hand side in the series decrease the hydrophobic effect, and thus help maintain the proteins in solution.

Polar organic solvents such as ethanol promote the precipitation of proteins due to the decrease in water activity in the solution as the water is replaced by organic solvent. They have been widely used as precipitation agents, especially in the fractionation of serum proteins. The following five variables are usually kept under control: concentration of organic solvent, protein concentration, pH, ionic strength, and temperature (17). Low temperature during the precipitation operations is often necessary to avoid protein denaturation; the addition of an organic solvent decreases the freezing point of the solution and temperatures below 0°C can be used. In reversed phase chromatography, some proteins can be chromatographed in solutions that contain up to ~50% organic solvent, with retention of their biological activity.

Organic polymers function in a way similar to that of organic solvents. The most widely used polymer is polyethylene glycol (PEG), with an average molecular weight of either 6000 or 20,000. The main advantage of PEG over organic solvents is that it is more easily handled. It is unflammable, not poisonous, uncharged, and relatively unexpensive. Rather low concentrations are required (often less than 25%) to precipitate most proteins. One disadvantage is that high concentration solutions of PEG are viscous. PEG can also be difficult to remove from protein solutions. However, after dilution with buffer the viscosity decreases, and because the substance is uncharged, the solution may be applied directly to an ion exchange column to further separate the proteins, simultaneously removing the polymer.

pH adjustment has been used as a simple and cheap way to precipitate proteins. Proteins have their lowest solubility at their isoelectric point. This is sometimes used in serum fractionation and also in the purification of insulin.

Besides pH, another parameter that influences precipitation of proteins in salt solutions is temperature (see below). Keeping the salt concentration constant and varying the temperature is another way of fractionating a protein solution.

The salting out of a protein can be described by the equation

where S is the solubility of the protein in g/L of solution, B is an intercept constant, K is the salting out constant, and c is salt concentration in mol/L.

The value of B depends on the salt used, the pH, the temperature, and the protein itself; K depends on the salt used and the protein. It should be stressed that addition of a salt or another precipitating agent to a protein solution only decreases the solubility of the proteins. This is why a very dilute protein solution for precipitation may lead to low recovery, because a major part of the protein simply remains in solution. Reproducible results can only be achieved if all the parameters mentioned above, including the protein concentration, are kept constant.

Centrifugation is used routinely in the protein purification laboratory to recover precipitates. It can also be used to separate two immiscible liquid phases. Another application is density gradient centrifugation. Today this is used predominantly for the fractionation of subcellular particles and nucleic acids. An alternative is the use of liquid-liquid phase extraction, which seems to offer several advantages over the more classical methods.

1.3.2 Electrophoresis

Electrophoresis in free solution or in macroporous gels such as 1–2% agarose separates proteins mainly according to their net electric charge. Electrophoresis in gels such as polyacrylamide separates mainly according to the molecular size of the proteins.

Today, analytical gel electrophoresis requiring microgram amounts of proteins is an important tool in bioscience and biotechnology (see Chapter 15). Convenient methods for the extraction of proteins after electrophoresis have been developed, in particular protein blotting (see Chapter 18), making the technique micropreparative. There are also many instances where a very small amount of protein is sufficient for the analysis of size and composition as well as the primary structure. Finally, there are cases where the starting material is extremely limited, such as protein extracts from small amounts of tissue (biopsies, etc.). In these cases, the protein “extract” might be just large enough for gel electrophoretic analysis.

Larger scale (milligrams to grams of protein) electrophoresis was an important method for the fractionation of protein extracts during the 1950s and early 1960s. It was carried out using columns packed with, for example, cellulose powder as a convection depressor, as in the “Porath column” (18). An innate limitation of preparative column electrophoresis is the joule heat developed during the course of the experiment. This means that the column diameter, if it is to allow sufficient cooling, should not exceed ~3 cm. Several hundred milligrams of protein can, however, be separated on such columns. Column zone electrophoresis has the advantage of allowing a precise description of the separation parameters involved and is, besides gel filtration, the mildest separation technique available for proteins. It can be recommended for special situations, but practical aspects and the excessive time required precludes its routine use. Methods for large and medium scale preparative electrophoresis have been developed, such as the flowing curtain electrophoresis of Hannig (19) and, more recently, the “Biostream” apparatus of Thomson (20).

Isoelectric focusing, the other main electrophoretic technique, separates proteins according to their isoelectric points (see Chapter 16). This technique gives very high resolution, but presents major difficulties as a preparative large or medium scale technique. Special equipment is required to allow cooling during the focusing. Proteins often precipitate at their isoelectric point, and this precipitate can contaminate the other bands when a vertical SephadexTM bed or column with sucrose gradient is used as an anticonvection medium in the focusing experiment. Modern equipment for preparative isoelectric focusing (21) avoids these problems by dividing the separation chamber into smaller compartments. Another solution is to carry out the isoelectric focusing in a horizontal trough of sedimented gel particles such as Sephadex (22). Here, precipitation in one zone will not disturb the other bands. On the other hand, the recovery of proteins is more tedious.

For routine preparative protein fractionation the electrophoretic techniques have become less important than chromatography. Ion exchange chromatography depends on parameters similar to those for electrophoresis. Chromatofocusing fractionates proteins largely according to their isoelectric points and would therefore appear to be a more convenient alternative to preparative isoelectric focusing.

1.3.3 Chromatography

Separation by chromatography depends on the differential partition of proteins between a stationary phase (the chromatographic medium or the adsorbent) and a mobile phase (the buffer solution). Normally, the stationary phase is packed into a vertical column of plastic, glass, or stainless steel, and the buffer is pumped through this column. An alternative is to stir the protein solution with the adsorbent, batchwise, and then pour the slurry onto an appropriate filter and make the washings and desorptions on the filter.

Column chromatography has proved to be an extremely efficient technique for the separation of proteins in biological extracts. Since the development of the first cellulose ion exchangers by Peterson and Sober (23) and of the first practical gel filtration media by Porath and Flodin (24, 25) a wide variety of adsorbents have been introduced that exploit various properties of the protein for the fractionation. The more important of these properties, together with the chromatographic method for which they dominate the separation, are as follows:

1. Size and shape (gel filtration/size exclusion chromatography, SEC).

2. Net charge and distribution of charged groups (ion exchange chromatography, IEC).

3. Isoelectric point (chromatofocusing, CF).

4. Hydrophobicity (hydrophobic interaction chromatography, HIC; reversed phase chromatography, RPC).

5. Metal binding (immobilized metal ion affinity chromatography, IMAC).

6. Content of exposed thiol groups (covalent chromatography, CC).

7. Biospecific affinities for ligands, inhibitors, receptors, antibodies, and so on (affinity chromatography, AC).

The methods often have very different requirements with regard to chromatographic conditions., including ionic strength, pH, and various additives such as detergents, reducing agents, and metals. By appropriate adjustment of the buffer composition, the conditions for adsorption and desorption of the desired protein can be optimized. It should be stressed that the result of a particular chromatographic separation often depends on more than one parameter. In IEC, the charge interaction is the dominant parameter, but molecular weight and hydrophobic effects can also contribute to some degree, depending on the experimental conditions and type of solid phase used. In recent years the concept of multimodal, or mixed-mode adsorption chromatography, has received an increasing amount of attention, with several new products emerging on the market (see Chapter 4 for more detailed information).

Highly specific methods, such as those based on bioaffinity (e.g., antibody–antigen interaction) or those based on the use of in vivo fused tags such as (His)6 or glutathione-S-transferase (GST), do in some cases give a highly pure protein in a single step. Normally, however, several chromatographic methods have to be combined in order to achieve maximal purification of a protein from a crude biological extract. With the wide variety of chromatographic media available today, in combination with a modern computerized chromatography system, adequate purification can normally be achieved within a few days to a couple of weeks.

In recent years, columns containing a continuous, homogeneously porous solid phase have become available. See Chapters 2 and 9 for more information about these so-called monolithic column types. As in membrane adsorption techniques, the main advantage is the considerably reduced diffusion restriction, allowing high efficiency and also high flow rates. The main disadvantage of both these techniques is the concomitant smaller surface area per unit adsorption medium volume, which will restrict the nominal column binding capacity.

All of the chromatographic methods mentioned above are treated in Part II of this book, which begins with a general description of the concepts used in protein chromatography.

1.3.4 Expanded Bed Adsorption

The problem of removing cells and cell debris from large volumes of whole cell cultures or cell homogenates has encouraged the development of technologies for the direct adsorption of target molecules from such feed stocks. In a fiuidized bed, the adsorbent particles are subject to an upward flow of liquid that keeps them separated from each other. The resulting increased voidage allows the unhindered passage of cells and cell debris. In a typical fiuidized bed there is a total mixing of particles and sample in the reactor, leading to incomplete adsorption of the target molecules unless the feed stock is recycled. Expanded bed adsorption is a special case of fiuidized bed adsorption (26), and is primarily applied in a pilot- or production-scale environment (26–29).

1.3.5 Membrane Adsorption

The main argument for utilizing modified membranes as media for protein adsorption is to solve the problem of mass transport restriction in standard chromatography due to the slow diffusion of proteins in the pores of the large gel particles. In membranes, most pores allow convective flow, and the mass transport resistance is therefore minimized to film diffusion at the membrane matrix surface. The result is a more efficient adsorption–desorption cycle of target solutes, allowing considerably higher flow rates and thus considerably shorter separation times. The area has been reviewed by Thömmes and Kula (30). See Chapter 12 for more data regarding membrane separation.

1.4 FRACTIONATION STRATEGIES

1.4.1 Introductory Comments

Before attempting to design a purification protocol for a particular protein, as much information as possible should be collected about the characteristics of that protein and preferably also about the properties of the most important known impurities. Useful data include approximate molecular weight and pI, degree of hydrophobicity, presence of carbohydrate (glycoprotein) or free -SH. Some of this information might be obtained already on a DNA level, if nucleotide sequence data are available, but is otherwise often collected easily by preliminary trials using crude extracts.

Criteria with regard to the stability of the protein to be purified should be established. Important parameters affecting structure are temperature, pH, organic solvents, oxygen (air), heavy metals, and mechanical shear. Special concern should be addressed to the risk of proteolytic degradation. Finally, it is the amount of protein to be purified per batch, and the required degree of purity, that to a high degree governs the techniques and methods used in the purification process.

According to a study of 100 published successful protein purification procedures (31), the average number of steps in a purification process is four. Very seldom can a protein be obtained in pure form from a single chromatographic procedure, even when this is based on a unique biospecificity. In addition to the purification steps there is often a need for concentrations and sometimes changes of buffers by dialysis or membrane ultrafiltration.

The preparation scheme can be described as consisting of three stages:

1. The preliminary or initial fractionation stage (often called the capturing stage).

2. The intermediate purification stage.

3. The final polishing stage.

The purpose of the initial stage is to obtain a stable, particle-free solution suitable for chromatography. This is achieved by clarification, coarse fractionation, and concentration of the protein extract. The purpose of the final stage is to remove aggregates and degradation products and to prepare the protein solution for the final formulation of the purified protein.

Sometimes one or two of these stages coincide. An initial ion exchange adsorption step can thus serve as a preliminary fractionation applied directly to the protein extract, or a gel filtration can give a product that is suitable as a final product, However, as the purposes of the three stages are different it is useful to discuss them separately.

The design of the preparation scheme will differ depending on the material at hand and the purpose of the purification. If the starting material is very precious, one should favor high yield over speed and convenience. In cases where several different proteins are to be extracted from a single starting material, this of course also affects the planning of the work. Finally, the final step is designed so that the product will be suitable for its purpose, which can vary. These aspects will be discussed below.

1.4.2 Initial Fractionation

There are many methods for the clarification of protein solutions. Extracts of fungal or plant origin often contain phenolic substances or other pigments. These can be removed by adsorption to diatomite (diatomaceous earth, Celite), either batchwise or on a short column. In order to prevent oxidation and miscoloration, small amounts of ascorbic acid can be added to the crude plant extract.

Similarly, lipid material can be removed either by centrifugation, as the lipids will float, and one thus needs to extract the protein solution from below, or by a chromatographic procedure. Lipids adsorb to a number of materials. Aerosil, a fused silica, has been used for the adsorption of lipids, but agarose is sometimes a simple choice.

Contamination with nucleic acids can, in some cases, especially when preparing proteins from bacteria, constitute a problem due to their high viscosity. The classical way to solve this problem is to precipitate the nucleic acids. Streptomycin sulfate and polyethylenimine have been used as precipitants, as have protamine sulfate and manganese salts (32). Another way to solve the problem is to add nucleases, which cut the nucleic acids into smaller pieces, thereby reducing the viscosity. Another problem with nucleic acids or degradation products of nucleic acids is that, due to their low isoelectric points, they still are negatively charged at low pH. Anion exchangers strongly adsorb nucleic acids and are thus difficult to regenerate. The solution to this problem can in some cases be to perform two consecutive adsorption steps. The first is executed at a pH below the pI of the target protein, thus preventing it from binding to the ion exchanger. Often, a fairly small amount of the ion exchanger is required in this step, which is why it is economically motivated to discard the contaminated gel. In the second step using the same anion exchanger, the pH is increased to a value above the pI of the target protein, resulting in binding and subsequent elution using either a stepwise or continuous salt gradient.

1.4.2.1 Clarification by Centrifugation and/or Microfiltration The clarification of any cell homogenate is usually no problem on a laboratory scale, where refrigerated high speed centrifuges operating at speeds from 20,000 rpm to 75,000 rpm, generating from ~40,000g to ~500,000g can be used. A useful review of centrifugation and centrifuges in preparative biochemistry is found in Reference 33. As a complement to centrifugation, in recent years, tangential or cross-flow microfiltration has received increased attention, especially for large-scale applications. For a review of the advantages of cross-flow microfiltration we suggest Reference 34. The area is also treated in more detail in Chapter 12.

1.4.2.2 Ultrafiltration Ultrafiltration has become a widely used technique in preparative biochemistry. Ultrafiltration membranes are available with different cut-off limits for separation of molecules from 1 kDa up to 300 kDa. The method is excellent for the separation of salts and other small molecules from a protein fraction with higher molecular weight and at the same time can effect a concentration of the proteins. The process is gentle, fast, and inexpensive. Ultrafiltration is treated in more detail in Chapter 12.

1.4.2.3 Precipitation Crude extracts are seldom suitable for direct application to chromatographic columns. Preparative differential centrifugation seldom results in a sufficiently clear solution. This is one reason why it is often necessary to use other means for clarification that simultaneously concentrate the solution and remove most of the bulk proteins. Such an initial fractionation step should also result in the removal of proteases and membrane fragments that sometimes bind the protein of interest in the absence of detergents. The classical means is to make a fractional precipitation. Bulk proteins in the solution are first precipitated together with residual particulate matter, and then the protein of interest can be precipitated from the resulting supernatant solution. Sometimes the protein of interest is allowed to remain in the mother liquor solution for direct application to chromatographic columns, for example, hydrophobic interaction adsorption of proteins in ammonium sulfate solutions and IEC of proteins in PEG mother liquors. The most commonly used precipitating agents are listed in Table 1.5, together with some of their properties. A typical precipitation curve is shown in Figure 1.2.

Figure 1.2 Example of a precipitation curve, showing the amount of protein precipitated with a stepwise increase in ammonium sulfate concentration.

Of the various methods available for protein precipitation, the classical ammonium sulfate has some disadvantages. The resulting protein solution often needs to be dialyzed to obtain an ionic strength that allows IEC. This problem is avoided when using PEG. Organic solvents, in particular ethanol and acetone, often produce extremely fine powder-like precipitates that are difficult to centrifuge and handle. They have also often been shown to cause partial denaturation of proteins, which can, for example, prevent subsequent crystallization. This is why organic solvents are not recommended as first-choice precipitating agents.

1.4.2.4 Liquid-Liquid Phase Extraction A