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

696 Pages·2000·23.787 MB·English

by  Simon M.I., Thorner J., Emr S.D.

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,