Protein Biotechnology

Biological Methods

Protein Biotechnology: /so/a tion, Characterization, and Stabilization, edlted by Felix Franks, 1993

Enzymatic Analysis: A Pracfical Guide by janet V. Passonneau and Oliver H. Lowry, 1993

Characterization of Proteins, edi ted by Fe/ix Franks, 1988

Methods of Protein Microcharacterization: A Practical Handbook edited by John E. Shively, 1986

Techniques in Cell Cycle Analysis, edited by Joe W. Gray and Zbigniew Darzynkiewicz, 1986

Experiments in Molecular Biology, edited by Robert/. Slater, 1986

liquid Chromatography in Clinical Analysis, edited by Pokar M. Kabra and Laurence]. Mat-ton, 1981

Metal Carcinogenesis Testing: Principles and In Vitro Methods, by Max Costa, 1980

Protein Biotechnology Isolation, Characterization, and Stabilization

Edited by

Fe/ix Franks l pafra M, Cambridge, England

Humana Press, Totowa, New Jersey

0 1993 The Humana Press Inc 999 Rivervrew Drive, Suite 208 Totowa, New Jersey 07512

All rights reserved

No part of this book may be reproduced, stored in a retrieval system, or transmrtted In any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publrsher.

Printed in the United States of America. IO 9 8 7 6 5 4 3 2

Library of Congress Cataloging-in-Publication Data

Main entry under title:

Protein biotechnology : isolation, characterization, and stabilization / edited by Fekx Franks.

p. cm - (Brologcal methods) Includes Index. ISBN o-89603-230-2 1. Proteins-Biotechnology. I Franks, Felix II. Series [DNLM: 1. Proteins-analysis. 2. Proterns-physiology.

3. Biotechnology-methods. QU 55 P9653 19931 TP246.65.P76P72 1993 660’.63---dc20 DNLM/DLC for Library of Congress 93-3448

CIP

Preface

Proteins are the servants of life. They occur in all compo- nent parts of living organisms and are staggering in their func- tional variety, despite their chemical similarity. Even the simplest single-cell organism contains a thousand different pro- teins, fulfilling a wide range of life-supporting roles. Additions to the total number of known proteins are being made on an increasing scale through the discovery of mutant strains or their production by genetic manipulation.

The total international protein literature could fill a medium- sized building and is growing at an ever-increasing rate. The reader might be forgiven for asking whether yet another book on proteins, their properties, and functions can serve a useful purpose. An explanation of the origin of this book may serve as justification. The authors form the tutorial team for an inten- sive postexperience course on protein characterization orga- nized by the Center for Professional Advancement, East Brunswick, New Jersey, an educational foundation. The course was first mounted in Amsterdam in 1982 and has since been repeated several times, in both Amsterdam and the US, with participants from North America and most European countries.

In a predecessor to this book, emphasis was placed on the role of protein isolation in the food industry, because at the time this reflected the interests of most of the participants at the course. Today, isolated proteins for food use are extracted from yeasts, fungal sources, legumes, oilseeds, cereals, and leaves. Large-scale isolation methods have been developed and are operated on the basis of cost and efficiency. Any improvement

V

vi Preface

in the product is measured against the cost of achieving such an improvement, and this cost features largely in the market- ing equation along with other factors, such as the perceived (by the consumer) benefit of such an improvement. This latter atti- tude is of considerable importance in protein isolation technology.

In recent years interest in proteins has shifted away from food components, to their role in fighting disease and their uses as ana- lytical tools and biocatalysts. These major changes are reflected in the present book. It is a pleasure, however, to acknowledge the indirect contribution to this volume by two of the authors of the previous book. Some of the subject matter relating to food proteins and earlier discussed by Peter J. Lillford and Chester Myers has been incorporated, particularly in Chapters 2 and 5. We are also grateful to S. Fischer, a former tutor in our protein characterization course, for permitting us to use some material from his course notes in Chapter 2.

Probably the most important advances in recent years have been the developments in recombinant DNA technology that have led to massive commercial interest in the production (or creation) of highly specific proteins, such as interferon, hormones, and blood factors, and to the current boom in biotechnology. Here again the emphasis is on profitability (and a quick return on capital), so that allowable costs incurred in extraction and recovery must be deter- mined by the value of the product in the marketplace.

Market information on protein materials is closely guarded and hard to come by and, in any case, soon becomes out of date. We have attempted to include such economic aspects as were avail- able at the time of writing. The most comprehensive data refer to the production and marketing of enzymes.

Although much of the subject matter contained in this book can be found in other publications, its combination in one volume has probably never been attempted before. The viewpoint taken here is that methods developed for the isolation, purification, processability, analysis, stabilization, and characterization of pro- teins rely on our present knowledge of their structures, stabilities, and performance under a variety of physical and chemical condi-

Preface vii

tions. These methods are described and related to economic, clini- cal, and technological criteria. In other words, we are here con- cerned with the methodologies of relevance to the “protein business.” Protein-converting is a young, but rapidly growing, industry; some of the quantitative estimates here represented are therefore based on extrapolations of presently available data, which may turn out to be wide off the mark, but this is surely the general nature of industrial and economic forecasts.

In any discussion of the technology of protein isolation and processing from a business perspective, the value of the final prod- uct needs to be considered, since this will probably determine the method chosen for the extraction and purification of the protein. Another factor that needs to be considered is the scale of the operation.

An attempt is made to provide a unified approach to the problems of protein characterization, stability, analysis, isola- tion, and processing. Differences are drawn between in vivo and in vitro methods of characterization and between analyti- cal procedures that are appropriate to the quantities of mate- rial to be handled. Certain basic physical and chemical principles exist, whatever the scale of the operation, and these principles are summarized in the opening chapters.

Finally, a word of warning: Historically, much of our knowl- edge regarding protein structure, stability, and performance has been derived from in vitro studies on isolated proteins in dilute solution, acting on isolated substrates. An increasing number of such proteins is becoming available in bottled form, obtainable from laboratory suppliers, a prime example being lysozyme, on which much of the pioneering work was done. Whatever useful informa- tion can be obtained about lysozyme from model studies in aque- ous solutions of the enzyme, it must be remembered that, in vivo, a lysozyme molecule is most unlikely ever to “see” another lysozyme molecule. It is questionable, therefore, whether a study of the protein-protein interactions that are responsible for the crys- tallization of globular molecules has any relevance to physiologi- cal situations. On the other hand, such interactions are of great importance in isolation and purification technology.

. . . VIII Preface

We emphasize, therefore, that despite some common physico- chemical principles, clear distinctions must be drawn between physiological and technological function and performance and between results obtained from in vivo and in vitro characteriza- tion and analysis.

Felix Franks Cambridge, England

January 1993

Contents

One

Two

Three

Four

Five

Six

Seven

Preface l v

Contributors l xi

Proteins: Description and Classification l 1 Felix Franks

In Vitro Characterization:

Economics and Technology l 21 Felix Franks

Analytical Chromatography

of Amino Acids, Peptides, and Proteins l 49

James L. Dwyer

Internal Structure and Organization:

Relationship to Function l 91

Felix Franks

Solution Properties of Proteins l 133

Felix Franks

Posttranslational Processing of Proteins l 191

Johannes M. F. G. Aerts and And& W. Schram

Protein Fragmentation l 237

Linda A. Fothergill-Gilmore

ix

X Contents

Eight

Nine

Ten

Eleven

Twelve

Thirteen

Fourteen

Fifteen

Peptide Sequence Determination l 251 Linda A. Fothergill-Gilmore

Electrophoretic Techniques of Analysis and Isolation l 313 James L. Dwyer

Production and Application of Polyclonal and Monoclonal Antibodies l 365 Get-t Van Duijn and Andre W. Schram

Conformational Stability of Proteins l 395 Fe/ix Franks

Protein Hydration l 437 Felix Franks

Recombinant Protein Technology l 467 Linda A. Fothergill-Gilmore

Storage Stabilization of Proteins l 489 Felix Franks

Process Purification l 533 James L. Dwyer

Index l 573

Contributors

Johannes F. M. G. Aerts l University of Amsterdam,

Amsterdam, the Netherlands

James L. Dwyer l Ventec Inc., Marlborough, MA

Linda A. Fothergill-Gilmore l University of Edinburgh,

Edinburgh, Scotland

Felix Franks l Pafra Ltd., Cambridge, England

Andre W. Schram l Zaadunie BV, Enkhuizen,

the Netherlands

Gert Van Duijn l Center for Phytotechnology, Leiden,

the Netherlands

xi