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Gene Expression Technology PDF

670 Pages·1991·11.759 MB·English
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Preface The articles in this volume were assembled to enable the reader to design effective strategies for the expression of cloned genes and cDNAs. More than a compilation of papers describing the multitude of techniques now available for expressing cloned genes, this volume provides a manual that should prove useful for solving the majority of expression problems one is likely to encounter. The four major expression systems commonly available to most inves- tigators are stressed: Escherichia coli, Bacillus subtilis, yeast, and mamma- lian cells. Each of these systems has its advantages and disadvantages, details of which are found in Chapter 1 and the strategic overviews for the four major sections of the volume. The papers in each of these sections provide many suggestions on how to proceed if initial expression levels are not sufficient. DAVID V. GOEDDEL vx Contributors to Volume 185 Article numbers are in parentheses following the names of contributors. snoitail~A detsil are current. SRAL N~lSMHARBA (13), Department of -DiB ANITSIRHC Y. CHEN (35, 37), Department of molecular Chemistry, Genentech Inc., Cell Genetics, Genentech Inc., South San South San Francisco, California 94080 ,ocsicnarF California 94080 PAULINA SABLAB (3), Departamento de Bio- ASSENAV MLOHSIHC (35, 37), Department of logia Molecular, Centro de Investigacion Cell Genetics, Genentech Inc., South San sobre Ingenieria Genbtica y Biotechnolo- ,ocsicnarF California 94080 gia, Universidad Nacional Autonoma de A. MARK NAG1C (30), Laboratory of -celoM Mexico, Cuernavaca, Morelos, Mexico ular Genetics, National Institutes of CLINTON E. BALLOU (36), Department of Health, Bethesda, Maryland 20892 Biochemistry and Molecular Biology, Uni- MAILLIW REGNEVELC (25), Department of versity of California at Berkeley, Berkeley, Molecular Biology, lmmunex Corpora- California 94720 tion, Seattle, Washington 98101 RAMA M. EJAGALEB (8), Division of Molec- DORIS COlT (28), Chiton Corporation, ular and Cell Biology, Lilly Research Lab- Emeryville, California 94608 ,seirotaro Indianapolis, Indiana 46285 ENNA LLEB (29), Zymo Genetics Inc., Seat- D. SNESUOC (27), Wellcome, Beckenham, Kent, England tle, Washington 98105 OCSICNARF RAVILOB (3), Departamento de HERMAN A. ED BOER (9), suealroG -arobaL Biologia Molecular, Centro de Investiga- ,seirot University of Leiden, 2300 RA Lei- cion sobre Ingenieria Genbtica y Biotech- den, The Netherlands nologia, Universidad Nacional Autonoma DANIEL DIMAIO (45), Department of de Mexico, Cuernavaca, Morelos, Mexico Human Genetics, Yale University School DAVID BOTSTEIN (23), Genentech Inc., of Medicine, New Haven, Connecticut South San Francisco, California 94080 06510 RALPH A. WAHSDARB (33), Department of SAMOHT F. EUHANOD (30), Department of Biological Chemistry, College of Medicine, Biology, Indiana University, Bloomington, University of California at Irvine, Irvine, Indiana 47405 California 92 717 WERDNA J. DORNER (44), Department of YNOHTNA J. EKARB (34), Chiron -aroproC Molecular and Cellular Genetics, Genetics tion, Emeryville, California 94608 Institute Inc., Cambridge, Massachusetts SEMAJ R. HCAORB (22), Department of Biol- 02140 ogy, Princeton University, Princeton, New DLANOD J. OKNEBWOD (35), Department of Jersey 08544 Molecular immunology, Genentech Inc., COLIN R. CAMPBELL (41), Department of South San Francisco, California 94080 Genetics, University of Illinois College of NHOJ W. FFRODNEBUD (6), Department of Medicine, Chicago, Illinois 60612 Biology, Brookhaven National Laboratory, DIVAD F. LEAHCIMRAC (16), Synergen Inc., ,notpU New York 11973 Boulder, Colorado 80303 NHOJ J. DUNN (6), Department of Biology, A. SREBMAHC (27), Department of Biochem- Brookhaven National Laboratory, Upton, istry, University of Oxford, Oxford OX1 New York 11973 3QU, England TTOCS D. RME (21), Division of Biology, CHUNG NAN CHANG (35), Protein Design California Institute of Technology, Pasa- Laboratories, Palo Alto, California 94304 dena, California 91125 xi xii SROTUBIRTNOC OT EMULOV 185 TINA YRREVEHCTE (26), Department ofFer- Biological Sciences, Carnegie Mellon Uni- mentation Research, Genentech Inc., ,ytisrev Pittsburgh, Pennsylvania 15213 South San Francisco, California 94080 RANDAL J. KAUFMAN (39, 42, 44), Depart- DAVID V. GOEDDEL (1), Molecular Biology ment of Molecular and Cellular Genetics, Department, Genentech Inc., South San Genetics Institute Inc., Cambridge, Mas- ,ocsicnarF California 94080 sachusetts 02140 LARRY GOLD (2, 7), Department of Molecu- YLREBMIK YASLEK (29), Department of -DiB lar, Cellular and Developmental Biology, chemistry, University of British Columbia, University of Colorado at Boulder, Vancouver, British Columbia V6K 157, Boulder, Colorado 80309 Canada NASUS NAMSETTOG ( 11 ,) Laboratory of Mo- RICHARD L. KENDALL (33), Department of lecular Biology, National Cancer Institute, Biological Chemistry, College of Medicine, National Institutes of Health, Bethesda, University of California at lrvine, Irvine, Maryland 20892 California 92717 JAY D. GRALLA (4), Department of Chemis- WAYNE A. KEOWN (41), Department of -eG try and Biochemistry and the Molecular netics, University of Illinois College of Biology Institute, University of California Medicine, Chicago, Illinois 60612 at Los Angeles, West Los Angeles, -rofilaC nia 90024 A. J. KINGSMAN (27), Department of Bio- chemistry, University of Oxford, Oxford SICNARF J. GRANT (29), Zymo Genetics OX1 3QU, England Inc., Seattle, Washington 98105 ROBERT HAMILTON (35), Department of S. M. NAMSGNaK (27), Department of -DiB Fermentation Research and Process De- chemistry, University of Oxford, Oxford OX1 3QU, England velopment, Genentech Inc., South San Francisco, California 94080 TADAHIKO KOHNO (16), Synergen Inc., Boulder, Colorado 80303 DENNIS J. HENNER (5, ,71 20), Department of Genetics, Genentech Inc., South San MAILLIW J. KOHR (35), Department of Pro- ,ocsicnarF California 94080 tein Chemistry, Genentech Inc., South San RONALD A. HITZEMAN (35, 37), Depart- ,ocsicnarF California 94080 ment of Cell Genetics, Genentech Inc., MICHAEL KRIEGLER (40), Cetus Corpora- South San Francisco, California 94080 tion, Emeryville, California 94608 NYRHTAK J. HOFMANN (24), Department of RAJU S. ITAPALREHCUK (41), Department of Virus and Cell Biology, Merck Sharp and Genetics, University of Illinois College of Dohme Research Laboratories, West Medicine, Chicago, Illinois 60612 Point, Pennsylvania 19486 STUART F. J. LE GRICE (18), Department of SEMAJ E. HOPPER (24), Department of Bio- Infectious Diseases, Case Western Reserve logical Chemistry, The Milton S, Hershey University School of Medicine, Cleveland, Medical Center, Pennsylvania State Uni- Ohio 44106 ,ytisrev Hershey, Pennsylvania 17033 ARTHUR D. NOSNIVEL (38), Department of BRUCE H. HORWITZ (45), Department of Cell Genetics, Genentech Inc., South San Human Genetics, Yale University School ,ocsicnarF California 94080 of Medicine, New Haven, Connecticut CntmG LIU (35), Department of Protein 06510 Chemistry, Genentech Inc., South San ANNA S. HUI (9), Department of Pharmacol- Francisco, California 94080 ogy and Cell Biophysics, University of Cin- HONG MA (23), Division of Biology, -rofilaC cinnati, Cincinnati, Ohio 45267 nia Institute of Technology, Pasadena, HTEBAZILE W. JONES (31), Department of California 91125 CONTRIBUTORS TO VOLUME 185 xiii VIVIAN L. MACKAY (29), Zymo Genetics lecular and Cell Biology, Lilly Research Inc., Seattle, Washington 98105 Laboratories, Indianapolis, Indiana 46285 JENNIE P. MATHER (43), Department of Cell RONALD G. SCrtONER (8), Division of Mo- Culture Research and Development, Gen- lecular and Cell Biology, Lilly Research entech Inc., South San Francisco, Califor- Laboratories, Indianapolis, Indiana 46285 nia 94080 LOREN D. SCHULTZ (24), Department of SAMOT MOKS (12), Department of Biochem- Virus and Cell Biology, Merck Sharp & istry, The Royal Institute of ,ygolonhceT Dohme Research Laboratories, West S-100 44 Stockholm, Sweden Point, Pennsylvania 19486 ECNERWAL M. MYLIN (24), Department of ANDRZEJ Z. IKSWElZDELS (29), Zymo -eG Biological Chemistry, The Milton S. Her- netics Inc., Seattle, Washington 98105 shey Medical Center, Pennsylvania State SAMOHT J. YVAHLIS (15), Department of Bi- ,ytisrevinU Hershey, Pennsylvania 17033 ology, Princeton University, Princeton, AHTNASAV NAGARAJAN (19), Central Re- New Jersey 08544 search and Development Department, E. L NANCY J. NOSPMIS (35, 37), Department of du Pont de Nemours and Co., Wilmington, Cell Genetics, Genentech Inc., South San Delaware 19880 ,ocsicnarF California 94080 BJORN NILSSON (13), Department of Bio- ARJUN SINGH (35), Department of Cell -eG chemistry and Biotechnology, Royal Insti- netics, Genentech Inc., South San Fran- tute of Technology, S-100 44 Stockholm, ,ocsic California 94080 Sweden SAERDNA SOMMER (16), Biogrowth Inc., PETER O. OLINS (10), Biological Sciences Richmond, California 94806 Department, Monsanto Company, St. JOAN A. STADER (15), School of Basic Life Louis, Missouri 63198 Sciences, University of Missouri, Kansas ROGER PAI (35), Department of Product Re- City, Missouri 64110 ,yrevoc Genentech Inc., South San Fran- C. A. YAWNATS (27), Institute of Molecular ,ocsic California 94080 Medicine, Headington, Oxford, England AINIGRIV L. PRICE (25), Department of Mo- TIM SNRAETS (23), Department of Biochem- lecular Biology, Immunex Corporation, istry and Biophysics, University of -rofilaC Seattle, Washington 98101 nia, San Francisco, San Francisco, Cali- fornia 94143 SrIAUKAT H. RANGWALA (10), Biological Sciences Department, Monsanto Com- GARY D. OMROTS (7), Department of Molec- pany, St. Louis, Missouri 63198 ,ralu Cellular and Developmental Biology, University of Colorado at Boulder, MARK E. RENZ (35), Department of -leveD Boulder, Colorado 80309 opmental Biology, Genentech Inc., South San Francisco, California 94080 F. WILLIAM STUDIER (6), Department of Bi- ology, Brookhaven National Laboratory, ALAN B. ROSE (22), Department of ,ygoloiB ,notpU New York 11973 Princeton University, Princeton, New Jer- sey 08544 WAYNE E. TAYLOR (25), Department of Chemistry and Biochemistry, California ALAN H. GREBNESOR (6), Department of Bi- State University, Fullerton, California ology, Brookhaven National Laboratory, 92634 ,notpU New York 11973 AICIRTAP NOSLO-PMAKET (28), Chiron Cor- NEVETS GREBNESOR (28), Protos -aroproC poration, Emeryville, California 94608 tion, Emeryville, California 94608 ROnERT C. NOSPMOHT (16), Synergen lnc., ETTIGIRB E. RENOHCS (8), Division of Mo- Boulder, Colorado 80301 xiv CONTRIBUTORS TO VOLUME 185 SAIHTAM N~ILHU (12), Department of Bio- lac Chemistry, College of Medicine, Uni- chemistry, The Royal Institute of -lonhceT versity of ainrofilaC at lrvine, lrvine, -ilaC ,ygo S-IO0 44 Stockholm, Sweden fornia 92717 K~ITH D. NOSNIKLIW (32), Department of LEINAD G. ARUSNAY (5, 14), Department of ,yrtsimehcoiB Emory University School of Cell ,sciteneG Genentech Inc., South San Medicine, Atlanta, aigroeG 22303 ,ocsicnarF ainrofilaC 08049 M. WILSON (27), Delta Biotechnology Not- tingham, England ILRAC YIP (29), Zymo Genetics Inc., Seattle, notgnihsaW 50189 S1LRAM NOTGNIHTROW (25), Department of Biochemistry, University of ,notgnihsaW NOTLE T. YOUNG (25), Department of Bio- Seattle, Washington 59189 chemistry, University of Washington, Rvo ADAMAY (33), Department of -igoloiB Seattle, Washington 59189 1 SYSTEMS FOR HETEROLOGOUS GENE EXPRESSION 3 1 Systems rof Heterologous Gene Expression By DAVID .V GOEDDEL This volume concentrates on the four major expression systems that are commonly available to most investigators: Escherichia coli, Bacillus subtilis, yeast, and mammalian cells. Each of these systems has its advan- tages and disadvantages, some of which I briefly outline in this introduc- tory article. One expression system which is not covered in this volume is the baculovirus system in insect cells since it has been primarily developed by one group, and a detailed manual describing the system is available from that laboratory.t There are also examples in the literature of heterologous gene expression in a variety of other organisms, including Streptomyces, a number of fungi, pseudomonads, and others. Although these systems might become relatively more important in the future, we feel their utility has not at present been demonstrated broadly enough to recommend their routine use for heterologous protein production. In general, the expression of each cDNA or gene presents its own peculiar set of problems that must be overcome to achieve high-level expression. The synthesis of foreign proteins is still largely empirical. There is no set of hard-and-fast rules to follow. In fact, a particular protein is almost as likely to be the exception as it is to follow any set of rules. Keeping this caveat in mind, I will make some generalizations which I hope will aid the reader in selecting an initial expression system. Types of Proteins to Be Expressed For the purpose of gene expression in heterologous cells, proteins can be arbitrarily grouped into four broad classes. The first class covers small (less than -80 amino acids) peptides. These are most easily expressed as fusion proteins, usually in E. coll. The second class are polypeptides that are normally secreted proteins (e.g., enzymes, cytokines, hormones) and range in size from about 80 to 500 amino acids. This class of proteins is often the most straightforward to express (in all four systems) and secretion or direct expression should be considered the method of choice. In particu- lar, direct expression in E. coli has proved extremely effective for the subset of proteins in the 100-200 amino acid size range. A third class consists of very large (greater than about 500 amino acids) secreted proteins and cell i V. A. Lukow and M. D. Summers, Bio/Technology6, 47 (1988). thgirypoC © 0991 by cimedacA Press, Inc. METHODS IN ,YGOLOMYZNE VOL. 581 llA rights of reproduction in any form .devreser 4 INTRODUCTION 1 surface receptor proteins. Unless the protein of interest is of microbial origin, one generally has the most success with this class using a mamma- lian cell expression system. The fourth group encompasses all nonsecreted proteins larger than -80 amino acids. Many proteins fall into this class; however, relative to secreted proteins, much less work has been directed toward overexpression of proteins in this category. Therefore, the selection of an appropriate expression system should be based on the intended use for the protein (see below). For What Purpose Is the Expressed Protein Needed? Probably the most common reason for expressing a new gene or cDNA has been to verify that the correct sequence has, in fact, been isolated. If verification is all that is required, and if the protein being expressed is from a higher eukaryote, then transient expression in mammalian cells is the preferred expression route. Transient expression is not only easy to per- form, but also gives answers quickly and has a very high probability of yielding a biologically active protein. Historically, E. coli has also been used with great success for identification of mammalian proteins, often yielding biologically active protein, and, nearly always, immunoreactive material. For verification of microbial proteins, the generalization "make yeast proteins in yeast and bacterial proteins in bacteria" can be made. A relatively large amount of substantially pure protein is desirable for in vivo biological experiments or structural determination. In this case, it is worth taking the time and effort to evaluate more than one expression system to find the optimal one for that protein. Bacteria, yeast, and mam- malian cells have all been used to produce clinical grade proteins in large amounts. Escheriehia coli can be considered a good bet for the preparation of proteins, or portions thereof, to be used for the generation of antibodies. It is usually possible to express large quantities of antigen rapidly in E. coli, even though the overexpressed protein may not be correctly folded. In Section II of this volume there are articles on E. coli that outline, in addition to the old and still reliable trp vector systems, various novel methods for the direct and fusion expression of polypeptides suitable for antibody preparation. An increasingly common need for the expression of cloned genes is to obtain mutagenized protein for structure-function experiments (see 45 in this volume). The selection of the appropriate expression system for such studies is usually based on the ease of generating many defined mutants in an assayable form. Therefore, E. coli is an obvious first choice. 1 SYSTEMS FOR HETEROLOGOUS GENE EXPRESSION 5 However, if E. coli does not yield protein suitable for analysis, other systems must be examined. Obviously, if posttranslational modification contributes to the properties being studied, or if one is looking for pheno- typic changes in the host cells, the selection of an expression system should mimic the natural source of the protein as closely as possible. Escherichia coli Expression Systems Considerable lore has accumulated over the years concerning E. coil expression. It is often assumed that any protein can be produced in E. coli as long as it is not too small, too large, too hydrophobic, and does not contain too many cysteines. To some extent, these generalizations are correct; if one wants to express Factor VIII or a complex mammalian cell surface receptor containing 40 disulfides, E. coli is not the proper host. However, E. coli is a suitable, and often desirable host, for a large number of other expression needs. Escherichia coil systems for direct expression, fusion protein expression, and secretion, each of which has its particular advantages (and disadvantages), have been developed and refined. While it might be difficult to predict a priori the relative difficulty of purification for proteins produced by these three methods, there are usually other reasons for selecting one approach over the others. Fusion protein expression strategies ensure good translation initiation and often permit one to overcome the instability problems that can be encountered with small peptides. Furthermore, the high degree of certainty of expression by the fusion approach makes this a preferred method for generating immunogens. The fusion methods described in this volume also have purification advantages built into them, including insolubility of the fusion protein and purification protocols tailored to the fusion partner. Direct expression is usually the ideal method for E. coli production of a heterologous protein of 100-300 amino acids, assuming there is not an inordinate number of cysteines. The reducing environment present in E. coli does not permit cysteine- rich proteins to form the disulfide bonds required for proper conformation. This problem can sometimes be overcome by secretion into a more oxi- dized environment. However, secretion from E. coli is still largely a hit-or- miss proposition and is most likely to work with a naturally secreted protein (as opposed to trying to engineer the secretion of an intraceUular protein). There are many beneficial features to secretion: Problems with unwanted N-terminal methionines are avoided, proteins tend to fold better than during intracellular expression, and protein purification can often be simplified. 6 NOITCUDORTNI 1 Bacillus subtilis and Yeast Expression Systems Although B. subtilis and yeast are not usually thought of as first choices for heterologous gene expression, there are advantages of each that make them good candidates for specific expression needs. These advantages are discussed by Henner 17 and Emr 21 elsewhere in this volume. In particular, B. subtilis is a good choice for obtaining the secretion of a prokaryotic protein. Yeast offer many important advantages of both pro- karyotic and eukaryotic systems. Yeast grow rapidly, achieve high cell densities, and are able to make posttranslational modifications that bacte- rial systems are unable to perform. Many advances in yeast expression have helped place yeast on a more equal footing with E. coli as an initial choice for an expression system. Mammalian Cell noisserpxE smetsyS Progress in mammalian cell expression systems has been nothing short of astonishing in the last few years. Transient expression systems, which initially permitted the verification that a desired gene had been cloned, have become increasingly popular in a wide range ofcDNA cloning strate- gies. Furthermore, transient expression assays allow one to evaluate rapidly the relative merits of different vectors for subsequent use in stable expres- sion systems. Stable mammalian cell expression is more time-consuming than any of the other systems described in this volume; however, stable systems are essential for large-scale protein production in mammalian cells. The time and effort required for selection, amplification, and growth of stable cell lines are more than offset by the great versatility of mamma- lian cells in producing all of the different classes of protein described above; there are virtually no examples of proteins that cannot be made in at least detectable levels in mammalian cells. Synthetic DNA It si important to emphasize the benefits that can be gained by taking full advantage of the power of synthetic DNA. In making expression constructions, one should not be constrained by the DNA sequence or restriction sites present in the DNA to be expressed. Codon usage, poten- tial RNA secondary structure, and ribosome-binding-site sequences can be changed quite easily by the use of synthetic DNA, and the potential value of such alterations at improving expression should not be underestimated. It si worth considering as many variables as possible in designing an optimal expression vector for the gene of interest. If the existing sequences 1 SYSTEMS FOR HETEROLOGOUS GENE EXPRESSION 7 cannot be easily manipulated, replace and/or alter with synthetic DNA as much sequence as is necessary to achieve the "idealized" construction. Future Developments It is interesting to try to predict what surprises the next 5-10 years might hold for the heterologous expression of cloned genes. It is likely that there will be continued improvements made in the four major expression systems covered by this volume. Perhaps microbial hosts will be engineered so that they are able to perform specific mammalian posttranslational modifications such as the addition of complex sugars. It is even conceiv- able that each of the four host systems described here might be sufficient for any expression need, thereby eliminating the necessity of having access to more than one system. And finally, the recent demonstrations that heterologous proteins can be produced in the milk of transgenic animals makes it intriguing to speculate that even this technology might become an important component of the repertoire of a typical gene expression labora- tory.

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Most books are stored in the elastic cloud where traffic is expensive. For this reason, we have a limit on daily download.