Applications of Chimeric Genes and Hybrid Proteins: Cell Biology and Physiology

Preface The modem biologist takes almost for granted the rich repertoire of tools currently available for manipulating virtually any gene or protein of interest. Paramount among these operations is the construction of fusions. The tactic of generating gene fusions to facilitate analysis of gene expression has its origins in the work of Jacob and Monod more than 35 years ago. The fact that gene fusions can create functional chimeric proteins was demonstrated shortly thereafter. Since that time, the number of tricks for splicing or inserting into a gene product various markers, tags, antigenic epitopes, structural probes, and other elements has increased explosively. Hence, when we undertook assembling a volume on the applications of chimeric genes and hybrid proteins in modern biological research, we con- sidered the job a daunting task. To assist us with producing a coherent work, we first enlisted the aid of an Advisory Committee, consisting of Joe Falke, Stan Fields, Brian Seed, Tom Silhavy, and Roger Tsien. We benefited enormously from their ideas, suggestions, and breadth of knowledge. We are grateful to them all for their willingness to participate at the planning stage and for contributing excellent and highly pertinent articles. A large measure of the success of this project is due to the enthusiastic responses we received from nearly all of the prospective authors we ap- proached. Many contributors made additional suggestions, and quite a number contributed more than one article. Hence, it became clear early on that given the huge number of applications of gene fusion and hybrid protein technology-for studies of the regulation of gene expression, for lineage tracing, for protein purification and detection, for analysis of protein localization and dynamic movement, and a plethora of other uses-it would not be possible for us to cover this subject comprehensively in a single volume, but in the resulting three volumes, 326, 327, and 328. Volume 326 is devoted to methods useful for monitoring gene expres- sion, for facilitating protein purification, and for generating novel antigens and antibodies. Also in this volume is an introductory article describing the genesis of the concept of gene fusions and the early foundations of this whole approach. We would like to express our special appreciation to Jon Beckwith for preparing this historical overview. Jon’s description is particularly illuminating because he was among the first to exploit gene and protein fusions. Moreover, over the years, he and his colleagues have xvii xv111 PREFACE continued to develop the methodology that has propelled the use of fusion- based techniques from bacteria to eukaryotic organisms. Volume 327 is focused on procedures for tagging proteins for immunodetection, for using chimeric proteins for cytological purposes, especially the analysis of mem- brane proteins and intracellular protein trafficking, and for monitoring and manipulating various aspects of cell signaling and cell physiology. Included in this volume is a rather extensive section on the green fluorescent protein (GFP) that deals with applications not covered in Volume 302. Volume 328 describes protocols for using hybrid genes and proteins to identify and analyze protein-protein and protein-nucleic interactions, for mapping molecular recognition domains, for directed molecular evolution, and for functional genomics. We want to take this opportunity to thank again all the authors who generously contributed and whose conscientious efforts to maintain the high standards of the Methods in Enzymology series will make these volumes of practical use to a broad spectrum of investigators for many years to come. We have to admit, however, that, despite our best efforts, we could not include each and every method that involves the use of a gene fusion or a hybrid protein. In part, our task was a bit like trying to bottle smoke because brilliant new methods that exploit the fundamental strategy of using a chimeric gene or protein are being devised and published daily. We hope, however, that we have been able to capture many of the most salient and generally applicable procedures. Nonetheless, we take full responsibility for any oversights or omissions, and apologize to any researcher whose method was overlooked. Finally, we would especially like to acknowledge the expert assistance of Joyce Kato at Caltech, whose administrative skills were essential in organizing these books. JERJZMYTHORNER SCO?T D. EMR JOHN N. ABELSON Contributors to Volume 327 Article numbers are in parentheses following the names of contributors Affiliations listed are current. STEPHEN R. ADAMS (39, 40) Department of of Pennsylvania School of Medicine, Phila- Pharmacology and Howard Hughes Medi- delphia, Pennsylvania 19104-6160 cal Institute, University of California, San SHA~N BURGESS (ll), Center for Cancer Re- Diego, La Jolla, California 92093 search, Massachusetts Institute of Technol- THOMAS R. ANDERSON(~), Covance Research ogy, Cambridge, Massachusetts 02139 Products, Inc., Richmond, California 94804 JANICE E. Buss (26), Department of Biochem- V. ANDREEVA (28) Engelhardt Institute of istry and Biophysics, Iowa State University, Molecular Biology, Russian Academy of Ames, Iowa 50011 Sciences, Moscow 117984, Russia CONSTANCE L. CEPKO (lo), Department of BRIGITTE ANGRES (7), Clontech Laboratories, Genetics, Harvard Medical School and Inc., Palo Alto, California 94303 Howard Hughes Medical Institute, Boston, CHRISTOPHER AUSTIN (lo), Merck Research Massachusetts 02115 Laboratories, West Point, Pennsylvania RAY CHANG (34), Affymax Research Institute, I9486 Palo Alto, California 94304-1218 UDO BARON (30) Zentrum fiir Molekulare NEIL W. CHARTERS (20), Department of Biologie, Universitiit Heidelberg, Heidel- Molecular and Cell Biology, University of berg D-69120, Germany California at Berkeley, Berkeley, California JON BECKWITH (12), Department of Micro- 94720 biology and Molecular Genetics, Harvard HWAI-JONG CHENG (2, 15), Howard Hughes Medical School, Boston, Massachusetts Medical Institute and Department of Anat- 02115 omy, University of California, San Fran- S. BELLUM (28), Center for Molecular Medi- cisco, San Francisco, California 94143 cine, Maine Medical Center Research Insti- GEOFFREY J. CLARK (26), Department of Cell tute, South Portland, Maine 04106 and Cancer Biology, Division of Clinical CAROLYN R. BERTOZZI (20) Departments of Science, Medical Branch, National Cancer Chemistry, and Molecular and Cell Biology, Institute, Rockville, Maryland 20850-3300 University of California at Berkeley, Berke- DANIEL F. CUTLER (4) Medical Research ley, California 94720 Council Laboratory for Molecular Cell ANASTASIYA D. BLAGOVESHCHENSKAYA (4), Biology and Department of Biochemistry Medical Research Council Laboratory for and Molecular Biology, University College Molecular Cell Biology and Department of London, London WCIE 6BT, England, Biochemistry and Molecular Biology, Uni- United Kingdom versity College London, London WClE TAMARA DARSOW (8) Department of Biol- 6BT, England, United Kingdom ogy, University of California, San Diego, HERMANN BUJARD (30), Zentrum fur Mo- La Jolla, California 92093-0668 lekulare Biologie, Universitiit Heidelberg, CHANNING J. DER (26) Department of Phar- Heidelberg D-69120, Germany macology, Lineberger Comprehensive Can- CHRISTOPHER G. BURD (S), Department of cer Center, University of North Carolina at Cell and Developmental Biology and Insti- Chapel Hill, Chapel Hill, North Carolina tute for Human Gene Therapy, University 27599 Xi xii CONTRIBUTORS TO VOLUME 327 SCOTT D. EMR (8), Howard Hughes Medical KORET HIRSCHBERG (6), Cell Biology and Institute and School of Medicine, University Metabolism Branch, National Institute of of California, San Diego, La Jolla, Califor- Child Health and Human Development, nia 92093-0668 National Institutes of Health, Bethesda, Maryland 20892-5430 MICHAEL A. FARRAR (31), Merck Research Laboratories, Rahway, New Jersey 0706% KNUT HOLTHOFF (38), Department of Bio- 0900 logical Sciences, Columbia University, New York, New York 10027 JOHN D. FAYEN (27), Department of Pa- thology, Case Western Reserve University, B. DIANE HOPKINS (9), Department of Molec- ular Biology, Vanderbilt University, Nash- Cleveland, Ohio 44106 ville, Tennessee 37235 DAVID A. FELDHEIM (2), Department of Cell COLIN HOPKINS (3), Medical Research Coun- Biology, Harvard Medical School, Boston, cil Laboratory for Molecular Cell Biology, Massachusetts 02115 University College London, London WC1 E SHAWN FIELDS-BERRY (lo), Department of 6BT, England, United Kingdom Genetics, Harvard Medical School and NANCY HOPKINS (ll), Biology Department Howard Hughes Medical Institute, Boston, and Center for Cancer Research, Massachu- Massachusetts 02115 setts Institute of Technology, Cambridge, JOHN G. FLANAGAN (2, 1.5) Department of Massachusetts 02139 Cell Biology and Program in Neuroscience, BRYAN A. IRVING (16) Department of Micro- Harvard Medical School, Boston, Massa- biology and Immunology, University of chusetts 02115 California, San Francisco, San Francisco, CHRISTIAN E. FRITZE (l), Covance Re- California 94143-0414 search Products, Inc., Richmond, Califor- EHUD Y. ISACOFF (19), Department of Mo- nia 94804-4609 lecular and Cell Biology, University of CLARE FWI-LYR (3), Medical Research Council California at Berkeley, Berkeley, Cali- Laboratory for Molecular Cell Biology, fornia 94720-3200 University College London, London WC1 E LARA IZOTOVA (42), Department of Molecu- 6BT England, United Kingdom lar Genetics and Microbiology, University ADBLE GIBSON (3), Medical Research Council of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Pis- Laboratory for Molecular Cell Biology, cataway, New Jersey 08854-563.5 University College London, London WClE 6BT, England, United Kingdom CHRISTINA L. JACOBS (20) Departments of Chemistry, and Molecular and Cell Biology, JEFFREY GOLDEN (lo), Department of Pathol- University of California at Berkeley, Berke- ogy, Children’s Hospital of Philadelphia, ley, California 94720 Philadelphia, Pennsylvania 19104 JAY JONES (40), Aurora Biosciences Corpora- TODD R. GRAHAM (9), Department of Molec- tion, San Diego, California 92121 ular Biology, Vanderbilt University, Nash- ville, Tennessee 37235 STEVEN R. KAIN (7,37), Cellomics, Inc., Palo Alto, California 94301 GISELE GREEN (7), Clontech Laboratories, HEIKE KREBBER (22), Institut fiir Molekular- Inc., Palo Alto, California 94303 biologie und Tumorforschung, Philipps- B. ALBERT GRIFFIN (40), Aurora Biosciences Universitiit Marburg, 35033 Marburg, Corporation, San Diego, California 92121 Germany MITSUHARU HA-RORI (2), Department of Cell MARKKU S. KULOMAA (39), Department Biology, Harvard Medical School, Boston, of Biology, University of Jyvaskyla, FIN Massachusetts 02115 40351, Jyvaskyla, Finland . . . CONTRIBUTORS TO VOLUME 327 Xl11 M. LANDRISCINA (28) Center for Molecular LARRY C. MATHEAKIS (34), Afimax Re- Medicine, Maine Medical Center Research search Institute, Palo Alto, California Institute, South Portland, Maine 04106 94304-1218 JENNIFER A. LEEDS (12), Department of J. MICHAEL MCCAFFERY (39) Integrated Im- Microbiology and Molecular Genetics, Har- aging Center, Department of Biology, Johns vard Medical School, Boston, Massachu- Hopkins University, Baltimore, Maryland setts 02115 21218 WARREN J. LEONARD (17) Laboratory of M. EDWARD MEDOF (27) Departments of Molecular Immunology, National Heart, Pathology and Medicine, Case Western Lung, and Blood Institute, National Insti- Reserve University, Cleveland, Ohio 44106 tutes of Health, Bethesda, Maryland TOBIAS MEYER (36), Department of Pharma- 20892-1674 cology, Stanford University Medical School, JOHN LIN (lo), Department of Genetics, Har- Stanford, California 94305 vard Medical School and Howard Hughes GERO MIESENB~CK (38), Cellular Biochemis- Medical Institute, Boston, Massachusetts try and Biophysics Program, Memorial 02115 Sloan-Kettering Cancer Center, New York, New York 10021 LEI Lm (42) Department of Molecular Ge- netics and Microbiology, University of Med- REBECCA B. MILLER (38) Cellular Biochem- icine and Dentistry of New Jersey, Robert istry and Biophysics Program, Memorial Wood Johnson Medical School, Piscata- Sloan-Kettering Cancer Center, New York, New York 10021 way, New Jersey 08854-5635 ATXJSHI MIYAWAKI (35) Brain Research In- JENNIFER LIPPINCOTT-SCHWARTZ (6), Cell stitute, RIKEN, Wako City, Saitama 3.51- Biology and Metabolism Branch, National 0198, Japan Institute of Child Health and Human De- velopment, National Institutes of Health, HSIAO-PING H. MOORE (39), Department of Bethesda, Maryland 20892-5430 Molecular and Cell Biology, University of California at Berkeley, Berkeley, Califor- JUAN LLOPIS (39) Facultad de Medicina de nia 94720 Albacete, Universidad de Castilla-La Man- JOHN R. MURPHY (18) Department of Medi- cha, 02071 Albacete, Spain cine, Boston University School of Medicine, QIANG Lu (2), Department of Cell Biology, Boston, Massachusetts 02118 Harvard Medical School, Boston, Massa- AKIHIKO NAKIJNO (9) Molecular Membrane chusetts 02115 Biology Laboratory, RIKEN, Wako, Sai- TERRY E. MACHEN (39), Department of Mo- tama 351-0198 Japan lecular and Cell Biology, University of Cali- VALERIE NATALE (37), Clontech Labora- fornia at Berkeley, Berkeley, California tories, Inc., Palo Alto, California 94303 94720 DAVID A. NAUMAN (20), Departments of THOMAS MACIAG (28) Center for Molecular Chemistry, and Molecular and Cell Biology, Medicine, Maine Medical Center Research University of California at Berkeley, Berke- Institute, South Portland, Maine 04106 ley, California 94720 LARA K. MAHAL (20) Departments of Chem- ELENA OANCEA (36) Department of Neuro- istry, and Molecular and Cell Biology, Uni- biology, Childrens Hospital, Boston, Mas- versity of California at Berkeley, Berkeley, sachusetts 02115 California 94720 GREG ODORIZZI (8), Division of Cellular and YOSHIRO MARU (32), Department of Genet- Molecular Medicine, University of Califor- its, Institute of Medical Science, University nia and Howard Hughes Medical Institute, of Tokyo, Tokyo 108, Japan San Diego, La Jolla, California 92093-0668 xiv CONTRIBUTORS TO VOLUME 327 STEVEN H. OLSON (31), Merck Research Lab- ROSARIO RIZZ~TO (33) Department of En- oratories, Rahway, New Jersey 07065-0900 perimental and Diagnostic Medicine, Uni- versity of Ferrara, 44100 Ferrara, Italy HUGH R. B. PELHAM (21) MRC Laboratory of Molecular Biology, Cambridge CB2 VALERIE ROBERT (33), Department of Bio- 2QH, England, United Kingdom medical Sciences, CNR Centre of Biomem- branes, University of Padova, 35121 Pa- ROGER M. PERLMUTTER (31), Merck Re- dova, Ztaly search Laboratories, Rahway, New Jersey 07065-0900 ELIZABETH RYDER (lo), Department of Biol- ogy and Biotechnology, Worcester Poly- SIDNEY PESTKA (42), Department of Molecu- technic Institute, Worcester, Massachusetts lar Genetics and Microbiology, University 01609 of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Pis- KEN SATO (9) Molecular Membrane Biology cataway, New Jersey 08854-5635 Laboratory, RZKEN, Wako, Saitama 351- 0198 Japan ROBERT D. PHAIR (6) BioZnformatics Ser- CHRISTIAN SENGSTAG (13), ETH Zurich, Cen- vices, Rockville, Maryland 20854 ter for Teaching and Learning, Swiss Fed- DIDIER PICARD (29), Departement de Biologie eral Institute of Technology, CH-8092 Zu- Cellulaire, Universite de Geneve, Sciences rich, Switzerland ZZZ, 1211 Geneve 4, Switzerland EVE SH~NBROT (37), Clontech Laboratories, PAOLO PINTON (33) Department of Biomedi- Inc., Palo Alto, California 94303 cal Sciences, CNR Centre of Biomem- MICAH S. SIEGEL (19) Computation and Neu- branes, University of Padova, 35121 Pa- ral Systems Graduate Program, California dova, Italy Institute of Technology, Pasadena, Califor- TULLIO POZZAN (33) Department of Bio- nia 91Z25, and Department of Molecular medical Sciences, CNR Centre of Biomem- and Cell Biology, University of California at branes, University of Padova, 35121 Pa- Berkeley, Berkeley, California 94720-3200 dova, Italy PAMELA A. SILVER (22) Department of Bio- I. PRUDOVSKY (28), Center for Molecular logical Chemistry and Molecular Pharma- Medicine, Maine Medical Center Research cology, Harvard Medical School and The Institute, South Portland, Maine 04106 Dana Farber Cancer Institute, Boston, Mas- LAWRENCE A. QUILLIAM (26), Department of sachusetts 02115 Biochemistry and Molecular Biology, Zndi- D. SMALL (28) Center for Molecular Medi- ana University School of Medicine, Zndia- cine, Maine Medical Center Research Znsti- napolis, Indiana 46202-5122 lute, South Portland, Maine 04106 STEPHEN REES (34) Biological Chemistry R. SOLDI (28) Center for Molecular Medicine, Units, Glaxo Wellcome Research and De- Maine Medical Center Research Institute, velopment, Stevenage, Hertfordshire SGI South Portland, Maine 04106 2NY, England, United Kingdom COLLIN SPENCER (37) Rigel Corporation, MARILYN D. RESH (25), Cell Biology Pro- South San Francisco, California 94080 gram, Memorial Sloan-Kettering Cancer JENNY STABLES (34), Lead Discovery, Glaxo Center, New York, New York 10021 Wellcome Research and Development, Ste- GARY W. REUTHER (26) Department of Phar- venage, Hertfordshire SGI 2NY, England, macology, Lineberger Comprehensive Can- United Kingdom cer Center, University of North Carolina at IGOR STAGLJAR (14), Institute of Veterinary Chapel Hill, Chapel Hill, North Carolina Biochemistry, University of Zurich, 8057 27599 Zurich, Switzerland CONTRIBUTORS TO VOLUME 327 xv JANE STINCHCOMBE (3), Medical Research School of Medicine, Boston, Massachu- Council Laboratory for Molecular Cell Bi- setts 02118 ology, University College London, London ALEXANDER VARSHAVSKV (41), Division of WClE 6BT, England, United Kingdom Biology, 147-75, California Institute of STEPHAN TE HEESEN (14), ETH Zurich, Mi- Technology, Pasadena, California 91125 crobiology Institute, CH-8093 Zurich, Swit- KARSTEN WEIS (23), Department of Molec- zerland ular and Cell Biology, Division of Cell KEN TETER (39), Health Sciences Center, and Developmental Biology, University University of Colorado, Denver, Colo- of California at Berkeley, Berkeley, Cali- rado 80262 fornia 94720-3200 KOSTAS TOKATLIDIS (24), School of Bio- logical Sciences, University of Manchester, ARTHUR WEISS (16) Howard Hughes Medi- Manchester Ml3 9PT, England, United cal Institute and Departments of Medicine Kingdom and of Microbiology and Immunology, University of California, San Francisco, San VALERIA TOSELLO (33), Department of Bio- Francisco, California 94143 medical Sciences, CNR Centre of Biomem- branes, University of Padova, 35121 Pa- MINNIE M. Wu (39) Department of Molecu- dova, Italy lar and Cell Biology, University of Cali- ROGER Y. TSIEN (35, 39,40), Department of fornia at Berkeley, Berkeley, California Pharmacology and Howard Hughes Medi- 94720 cal Institute, University of California, San WEI WV (42), Department of Molecular Ge- Diego, La Jolla, California 92093 netics and Microbiology, University of Med- MARK L. TYKOCINSKI (U), Department of icine and Dentistry of New Jersey, Robert Pathology and Laboratory Medicine, Uni- Wood Johnson Medical School, Piscata- versity of Pennsylvania, Philadelphia, way, New Jersey 08854-5635 Pennsylvania 19104 KEVIN J. YAREMA (20), Departments of WOUTER VAN’T HOF (25) Pulmonary Re- Chemistry, and Molecular and Cell Biology, search Laboratories, Department of Medi- University of California at Berkeley, Berke- cine/lnstitute for Genetic Medicine, Weill ley, California 94720 Medical College of Cornell University, New York, New York 10021 RAFAEL YUSTE (38), Department of Biologi- cal Sciences, Columbia University, New PIERRE VANDERHAEGHEN (2), Department of Cell Biology, Harvard Medical School, York, New York 10027 Boston, Massachusetts 02115 SHIFANG ZHANG (38) Department of Bio- JOHANNA C. VANDERSPEK (18), Depart- logical Sciences, Columbia University, New ment of Medicine, Boston University York, New York 10027 111 EPITOPETAGGING 3 [l] Epitope Tagging: General Method for Tracking Recombinant Proteins By CHRISTIAN E. FRITZE and THOMAS R. ANDERSON Introduction Epitope tagging is a procedure whereby a short amino acid sequence recognized by a preexisting antibody is attached to a protein under study to allow its recognition by the antibody in a variety of in vitro or in vivo settings. Since its first use in 1987 by Munro and Pelham,l the epitope- tagging strategy has come to be widely utilized in molecular biology. As testimony to that fact, epitope tagging was employed in some manner in 30% (20 of 64) of the articles in a recent volume of the journal Cell (Vol- ume 98, July-October, 1999). Tagging a protein with an existing epitope is a simple procedure that allows researchers to readily purify or follow proteins through meaningful and revealing experiments quite promptly after expressing a cloned se- quence. This stands in sharp contrast to the several months that would otherwise be spent generating and characterizing antisera against the pro- tein itself. Highly specific antibodies and useful cloning vectors encoding epitope tags adjacent to cloning sites are readily available from commercial suppliers or erstwhile collaborators, adding to the ease of initiating such studies. The most obvious advantage of epitope tagging is that the time and expense associated with generating and characterizing antibodies against multiple proteins are obviated. However, epitope tagging offers a number of additional advantages. For example, because the tag would be missing from extracts of cells that are not expressing a tagged protein, negative controls are unequivocal. Experiments using antibodies against epitopes found in the native molecule cannot provide a comparable negative control. Similarly, epitope tagging can allow for tracking closely related proteins without fear of spurious results resulting from cross-reactive antibodies. The intracellular location of epitope-tagged proteins can be identified in immunofluorescence experiments in a similarly well-controlled manner, without fear of cross-reactivity with the endogenous protein. Because the experimenter has a choice of the tag insertion site in a protein, a site can be selected that is not likely to result in antibody interference with functional 1 S. Munro and H. R. Pelham, Cell 48,899 (1987). Copyright 0 2Mx) by Academic Press AU rights of reproduction in any form reserved. METHODS IN ENZYMOLOGY, VOL. 327 cu76-6?.79/00 $3O.M) 4 EPITOPE TAGS FOR IMMUNODETECTION ill sites in the molecule, for example, sites that might be the location of protein-protein interactions. Because the antigenic determinant of the epi- tope tag antibody is in each case defined by a specific peptide, that peptide can be used to elute fusion proteins in purification efforts, avoiding harsh conditions generally used in conventional affinity chromatography. Hence, tagging a protein immediately provides a straightforward purification strat- egy. Finally, the epitope-tagging approach may be particularly useful for discriminating among otherwise similar gene products that cannot be distin- guished with conventional antibodies. For example, epitope tagging permits discrimination of individual members of closely related protein families or the identification of in vitro-mutagenized variants in the context of endogenous wild-type protein. This chapter provides a brief summary of several common experimental procedures that make use of epitope tagging. An effort is made to suggest factors to be considered when designing or troubleshooting experiments in- volving epitope tagging. Interested readers are directed elsewhere for a de- scription of the historical development of epitope tagging or for a more exten- sive listing of bibliographic citations2 or to past reviews on this topic.3-5 General Considerations Choosing Tags The most commonly used epitope tags are outlined in Table I. In each case, monoclonal and polyclonal antibodies as well as cloning vectors are widely available. As the use of epitope tagging has become more wide- spread, a number of observations have been made that can occasionally suggest the preferred use of one tag over another. Several “pros and cons” are noted to help guide the researcher in choosing a tag appropriate to the application. The reader is cautioned, however, that each disadvantage noted in Table I has its exceptions. For example, whereas Table I indicates that the 9ElO antibody is a poor choice for experiments that involve immunopre- cipitation of tagged proteins, there are, of course, ample references in the literature to experiments in which immunoprecipitations were effectively accomplished with this antibody. A number of less commonly used tags are presented in Table II. These ’ http://www. babco.com/etagging.html; C. Fritze and T. Anderson, Biotechniques, in prepa- ration. 3 J. W. Jarvik and C. A. Telmer, Annu. Rev. Genet. 32,601 (1998). 4 Y. Shiio, M. Itoh, and J. Inoue, Methods Enzymol. 254, 497 (1995). 5 P. A. Kolodziej and R. A. Young, Methods Enzymol. 194,508 (1991). TABLE I COMMONLY USED EPITOPE TAGS Recognized Development of First use as Advantages Disadvantages antibody“ a tag” sequence Tag Highly specific second-generation Original 12CA5 antibody not op- 3 YPYDVPDYA 2 1, HA antibodies available timized for use in epitope tagging EQKLISEEDL Hybridoma line expressing the PElO monoclonal may not immu- 4 5 MYC PElO monoclonal obtainable noprecipitate reliably. Endoge- from the ATCC for use in nous c-myc expression inter- large-scale projects feres with use as epitope tag 6-8 FLAG DYKDDDK Epitope easily cleaved off of Detection by some antibodies re- 9 tagged protein after purifi- quires placement at the protein cation termini Tagged proteins can be purified Some commercially available anti- Polyhistidine HHHHHH on Ni2’ affinity matrix. Rare se- bodies require additional quence makes cross-reactivity amino acids to specify recog- with endogenous proteins un- nition likely a Key to references: (1) H. L. Niman, R. A. Houghten, L. E. Walker, R. A. Reisfeld, I. A. Wilson, J. M. Hogle, and R. A. Lemer, Proc. Natl. Acud. 80, 4949 (1983); (2) I. A. Wilson, H. L. Niman, R. A. Houghten, A. R. Cherenson, M. L. Connolly, and R. A. Lerner, Cell 37, 767 Sci. U.S.A. (1984); (3) J. Field, J. Nikawa, D. Broek, B. MacDonald, L. Rodgers, I. A. Wilson, R. A. Lemer, and M. Wigler, 2159 (1988); Mol. Cell. Biol. 8, (4) G. I. Evan, G. K. Lewis, G. Ramsay, and J. M. Bishop, Cell. 5,361O (1985); (5) S. Munro and H. R. Pelham, Cell 48,899 (1987); Mol. Biol. (6) K. S. Prickett, D. C. Amberg, and T. P. Hopp, 7,580 (1989); (7) B. L. Brizzard, R. G. Chubet, and D. L. Vizard, BioTechniques BioTechniques 16,730 (1994); (8) R. G. Chubet and B. L. Brizzard, 20,136 (1996); (9) T. P. Hopp, K. S. Prickett, V. L. Price, R. T. Libby, C. BioZ’echniques J. March, D. P. Cerretti, D. L. Urdal, and P. J. Conlon, 6, 1204 (1988); (10) E. Hochuli, W. Bannwarth, H. Dobeli, and R. Genti, BioTechniques 1321 (1988). Bio/Technology 6,