Applications of Chimeric Genes and Hybrid Proteins

614 Pages·2000·9.366 MB·English

by  Thorner J. (ed.), Emr S.D. (ed.), Abelson J.N. (ed.)

Preface
The modern 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
iiix
xiv ECAFERP
continued to develop the methodology that has propelled the use of fusion-
based techniques from bacteria to eukaryotic organisms. Volume 723 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.
YMEREJ RENROHT
Scott D. RME
NHOJ N. NOSLEBA
Contributors to Volume 326
Article numbers are in parentheses following the names of contributors.
Affiliations listed are current.
JON BECKWITH (1), Department of Microbiol- JOHN E. ,NANORC JR. (27), Departments of
ogy and Molecular ,sciteneG Harvard Med- Microbiology and Biochemistry, ytisrevinU
ical School, Boston, sttesuhcassaM 51120 of Illinois, ,anabrU Illinois 10816
JOSHUA A. BORNHORST (16), Department of MI DRALL G. LLUC (26), Avidity, L.L. C, -aelE
yrtsimehC and Biochemistry, University of nor Roosevelt Institute, Denver, Colorado
,odaroloC Boulder, Colorado 5120-90308 6O2O8
LISA RETSIERB (22), enegatartS Cloning Sys- NAYRB R. CULLEN (11), Howard Hughes
tems, La Jolla, California 73029 lacideM Institute and Department of -teneG
,sci Duke ytisrevinU Medical ,retneC Dur-
ANERI NIETSNORB (13), Tropix, Inc., PE -DiB
ham, North aniloraC 01772
,smetsys Bedford, sttesuhcassaM 03710
NAIRB D'EoN (13), Tropix, Inc., PE Biosys-
NOTYALC KCOLLUB (14), Department of -rahP ,smet Bedford, sttesuhcassaM 03710
macology, College of ,enicideM ytisrevinU
EROTAVLAS SITRAMED (29), Institute of -rahP
of ,ainrofilaC Irvine, California 79629
lacituecam ,secneicS Department of Applied
ANDREW CAMILLI (5), Department of -uceloM BioSciences, Swiss Federal Institute of
ral Biology and Microbiology, Tufts -inU ygolonhceT Zurich, CH-8057 Zurich, Swit-
ytisrev School of ,enicideM Boston, -assaM dnalrez
sttesuhc 11120
HTEBAZILE A. HTIMS-01SALB1D (21), sciteneG
SELRAHC R. ROTNAC (19), Center for Ad- ,etutitsnI ,egdirbmaC sttesuhcassaM 04120
decnav Biotechnology and Departments of
Roy H. Dol (25), Section of Molecular and
Biomedical gnireenignE and Pharmacology
ralulleC Biology, University of ,ainrofilaC
and Experimental Therapeutics, Boston
Davis, California 61659
,ytisrevinU Boston, Massachusetts 51220
and Sequenom, Inc., naS Diego, -rofilaC CHARLES F. EARHART (30), Section of -celoM
ular Genetics and Microbiology, ehT -inU
nia 12129
ytisrev of Texas ta Austin, Austin, Texas
NHOJ M. NIWGRIHC (20), Research ,ecivreS 5901-21787
Audie L. Murphy Memorial Veterans Ad-
HPLOD NOSFELLE (31), Department of -celoM
ministration Medical Center and Depart-
ular Microbiology and Immunology, Ore-
ments of Medicine and Biochemistry, Uni-
gon Health Sciences ,ytisrevinU ,dnaltroP
ytisrev of Texas Health Science Center at
Oregon 10279
naS Antonio, Texas 0093-92287
JOSEPH J. FALKE (16), Department of -simehC
GNOROAHS GNOHC (24), New England -DiB yrt and Biochemistry, University of Colo-
,sbal Inc., Beverly, sttesuhcassaM 51910 ,odar Boulder, Colorado 5120-90308
R. JOHN COLLIER (33), Department of -orciM ENIREHTAC ELLOYAF (32), ~tinU ed Biologie
biology and Molecular ,sciteneG Harvard sed snoitalug~R Immunitaires, CNRS URA
Medical School, Boston, Massachusetts ,5812 Institut Pasteur, Paris, Cedex ,51
51120 ecnarF
LISA A. COLLINS-RACIE (21), Genetics Insti- AILENROC NAMROG (14), DNA ,segdirB Inc.,
,etut Cambridge, sttesuhcassaM 04120 San ,ocsicnarF California 71149
ix
X CONTRIBUTORS TO VOLUME 326
PIERRE GUERMONPREZ (32), dtinU ed Biolo- COLIN MANOIL (3), Department of ,sciteneG
eig sed R~gulations Immunitaires, CNRS ytisrevinU of ,notgnihsaW ,elttaeS -gnihsaW
URA 2185, Institut Pasteur, Paris, Cedex ton 59189
,51 ecnarF
CHRIS MARTIN (13), Millennium evitciderP
NICHOLAS J. HAND (2), Department of ,enicideM ,egdirbmaC sttesuhcassaM 93120
Molecular Biology, Princeton ,ytisrevinU
DINA MARTIN (13), Tropix, Inc., PE Biosys-
,notecnirP New Jersey 44580
,snret Bedford, sttesuhcassaM 03710
FRED HEFFRON (6, 31), Department of
ROBERT A. MASTICO (34), Astbury ertneC for
Molecular Microbiology and Immunology,
larutcurtS Molecular Biology, ytisrevinU of
Oregon Health Sciences University, Port-
Leeds, Leeds LS2 9JT, United Kingdom
land, Oregon 10279
MARK McCORMICK (23), Novagen, Inc., Mad-
DANNY Q. HOANG (22), Stratagene Cloning
ison, Wisconsin 11735
Systems, La Jolla, California 73029
JOHN M. McCoY (21), Biogen, Inc., Cam-
PHILIPP HOLLIGER (28), MRC Laboratory of
,egdirb sttesuhcassaM 24120
Molecular Biology, Cambridge CB2 2QH
detinU Kingdom ROBERT C. MIERENDORF (23), Novagen, Inc.,
Madison, Wisconsin 11735
JOE HORECKA (7), Department of Molecular
Biology, NIBH, Tsukuba, Ibaraki 305- DARIO NERI (29), Institute of lacituecamrahP
6658 Japan Sciences, Department of Applied Bio-
ADRIAN HUBER (29), Institute of -uecamrahP ,secneicS Swiss laredeF Institute of -lonhceT
lacit ,secneicS Department of Applied Bio- ogy Zurich, CH-8057 Zurich, Switzerland
,secneicS Swiss laredeF Institute of -lonhceT FREDRIK NILSSON (29), Institute of Pharma-
ogy Zurich, CH-8057 Zurich, Switzerland ceutical Sciences, Department of Applied
SATOSHI INOUYE (12), Yokohama Research BioSciences, Swiss Federal Institute of
,retneC Chisso Corporation, Yokohama Technology Zurich, CH-8057 Zurich, Swit-
5068-632 Japan zerland
RAY JUDWARE (13), Tropix, Inc., PE Biosys- CORINNE E. M. OLESEN (13), Tropix, Inc., PE
,snret Bedford, sttesuhcassaM 03710 ,smetsysoiB Bedford, sttesuhcassaM 03710
GOUZEL KARIMOVA (32), dtinU ed Biochimie JAE-SEoN PARK (25), Sampyo Foods Co.,
,erialulleC CNRS URA 2185, Institut Ltd., Seoul ,040-231 Korea
,ruetsaP Paris, Cedex ,51 France DAVID PARKER (31), Department of -uceloM
NAA1TSIRHC KARREMAN (9), Institute of On- ral Microbiology and Immunology, Oregon
cological Chemistry, Heinrich Heine Uni- Health Sciences ,ytisrevinU Portland, Ore-
,ytisrev 40225 ,,frodlesseuD Germany gon 10279
DANIEL LADANT (32), Unit~ ed Biochimie HENRY PAULUS (24), Boston Biomedical Re-
,erialulleC CNRS URA 2185, Institut Pas- hcraes Institute, Watertown, sttesuhcassaM
,ruet Paris, Cedex ,51 ecnarF 9282-27420
EDWARD R. LAVALLIE (21), Genetics Insti- RONALD T. RA1NES (23), Departments of
,etut ,egdirbmaC sttesuhcassaM 04120 Biochemistry and Chemistry, University of
CLAUDE LECLERC (32), ~tinU ed Biologie sed ,nosidaM-nisnocsiW Madison, Wisconsin
Rdgulations Immunitaires, CNRS URA 60735
2185, lnstitut Pasteur, Paris, Cedex ,51 LAL1TA RAMAKRISHNAN (4), Department of
ecnarF Microbiology and Immunology, Stanford
BETTY LIU (13), Tropix, Inc., PE ,smetsysoiB ytisrevinU School of Medicine, Stanford,
Bedford, sttesuhcassaM 03710 ainrofilaC 4215-50349
ZHIJIAN LU (21), Genetics Institute, Cam- ENNYLEK E. REED (27), Department of Biol-
,egdirb sttesuhcassaM 04120 ,ygo Austin ,egelloC Sherman, Texas 09057
CONTRIBUTORS TO VOLUME 326 xi
DEEPALI SACHDEV (20), ytisrevinU of Minne- larutcurtS Molecular Biology, ytisrevinU of
sota Cancer Center, Minneapolis, Minne- Leeds, Leeds LS2 9JT, United Kingdom
sota 55455 NAI NOSNILMOT (28), MRC Laboratory of
EIIFOS REDA SALAMA (8), Microbia, Inc., Molecular Biology, Cambridge 2BC 2QH
,egdirbmaC sttesuhcassaM 93120 detinU Kingdom
TAKESHI SANO (19), Center for Molecular Im- A~yEs ULLMANN (32), Unit~ ed Biochimie
gniga sisongaiD and Therapy and Basic -icS ,erialulleC CNRS URA 2185, Institut
ence Laboratory, Department of ,ygoloidaR ,ruetsaP Paris, Cedex ,51 ecnarF
Beth Israel Deaconess Medical ,retneC Har- PETER TRUOCNALL1AV (22), enegatartS Clon-
vard Medical School, Boston, Massachu- ing Systems, La Jolla, California 73029
setts 51220 RAPHAEL H. VALDIVIA (4), Department of
PETER J. SCHATZ (26), Affymax Research In- Molecular dna Cell Biology, University of
,etutits Palo Alto, California 40349 ,ainrofilaC Berkeley, California 20749
THOMAS G. M. TD1MHCS (18), Institut far SUNA1RDA W. M. NAV RElD NEIDLEV (31), -eD
Bioanalytik GmbH, D-37079 ,negnittOG partment of Molecular Microbiology and
ynamreG Immunology, Oregon Health secneicS -inU
,ytisrev Portland, Oregon 10279
HAlE-SuN SHIN (25), Sampyo Foods Co., Ltd.,
Seoul ,040-231 Korea THOMAS R. VAN EIEIRBSOO (23), Novagen,
Inc., Madison, Wisconsin 11735
THOMAS J. SILHAVY (2), Department of
Molecular Biology, Princeton ,ytisrevinU ACSECNARF V1TI (29), Institute of -uecamrahP
lacit ,secneicS Department of Applied Bio-
,notecnirP New Jersey 44580
,secneicS Swiss laredeF etutitsnI of -lonhceT
ARNE SKERRA (18), Lehrstuhlfiir ehcsigoloiB
ygo Zurich, CH-8057 Zurich, Switzerland
,eimehC Technische tdtisrevinU Mfinchen,
JOHN C. VOVTA (13), Tropix, Inc., PE Biosys-
05358-D -gnisierF ,nahpetsnehieW Germany
tems, Bedford, sttesuhcassaM 03710
JAMES M. SLAUCH (5), Department of -orciM
MICAH J. YEILROW (6), Department of -celoM
biology, ytisrevinU of Illinois, ,anabrU Illi-
ular Microbiology and Immunology, Ore-
nois 10816
gon Health Sciences ,ytisrevinU ,dnaltroP
NElHPElTS SMALL (10), Department of ,ygoloiB Oregon 10279
New York University, New York, New
MING-QuN Xu (24), New England Biolabs,
York 30001
Inc., Beverly, sttesuhcassaM 51910
DONALD B. SMITH (17), Garden ,egattoC Yu-XIN YAN (13), Tropix, Inc., PE Biosys-
Clerkington, Haddington, East Lothian,
,smet Bedford, sttesuhcassaM 03710
,dnaltocS United Kingdom
REHPOTSIRHC C. ZAROZINSKI (33), Depart-
GEORGE F. SPRAGUE, JR. (7), Institute of ment of Microbiology dna Molecular -eG
Molecular Biology, University of ,nogerO ,sciten Harvard Medical School, Boston,
Eugene, Oregon 30479 sttesuhcassaM 51120
MICHAEL N. HCABNRATS (33), Department of CHAO-FENG ZHENG (22), enegatartS Cloning
Microbiology and Molecular ,sciteneG Har- Systems, La Jolla, California 73029
vard Medical School, Boston, Massachu- GREGOR ZLOKARNIK (15), Aurora Biosci-
sttes 51120 ences Corporation, naS Diego, ainrofilaC
PETER G. VEILKCOTS (34), Astbury Centre for 12129
1 3
THE ALL PURPOSE GENE FUSION
11 The All Purpose Gene Fusion
By JON BECKWITH
The biological revolution of recent years has derived its greatest impetus
from the development and utilization of a handful of techniques and ap-
proaches for manipulating DNA. These methods include, most prominently,
DNA cloning, DNA sequencing, the polymerase chain reaction, and gene
fusion. Given the advent of the first three technical developments only
during the past 25 years, one might have thought that the use of gene
fusions also appeared during this period. In fact, gene fusion as a method
for studying biological problems can be traced back to the earliest days of
molecular biology.
Many of the principles of the gene fusion approach appear in work on
one of the classical genetic systems of molecular biology, the rlI genes of
the Escherichia coli bacteriophage T4. In the late 1950s and early 1960s,
Seymour Benzer and colleagues charactered two adjacent but indepen-
dently transcribed genes, rlIA and rlIB, which constituted the rlI region.
In 1962, Champe and Benzer described an rlI mutation in which a deletion
(r1589) had removed all transcription and translation punctuation signals
between the two genes and, thus, fused them into a single transcriptional
and translational unit. 1 The deletion covered the sequences coding for the
carboxy terminus of the rlIA protein and for approximately 10% of the
amino terminus of the rlIB protein.
Despite the absence of a substantial portion of the B protein, the gene
fusion still exhibited B activity. This property of the r1589 deletion was to
provide a very important tool for understanding fundamental aspects of
the genetic code. These insights were made possible by the understanding
that missense mutations in the fusion that altered the A portion of the
hybrid rIIA-B protein would be unlikely to affect B function, whereas
mutations that caused termination of translation in the A portion would
simultaneously result in loss of B function. Benzer and Champe e found a
class of suppressible rlIA mutations that did have the effect of eliminating
rlIB activity when introduced into the r1589 deletion. These findings were
essential to the classification of these mutations (amber) as mutations that
cause protein chain termination. This was the first description of such
mutations and the recognition that special signals were involved in the
1 S. P. Champe and S. Benzer, J. Mol. Biol. 4, 288 (1962).
2 S. Benzer and S. P. Champe, Proc. Natl. Acad. Sci. U.S.A. 48, 1114 (1962).
Copyright © 2000 by Academic Press
All rights of reproduction in any form reserved.
METHODS IN ENZYMOI.OGY, VOL 623 0076-6879/00 $30.00
1
4 HISTORICAL OVERVIEW
chain termination process. At the same time, Crick and co-workers 3 were
characterizing a class of mutations that they suspected to be frameshifts.
A key step in their analysis was the demonstration that these mutations,
when introduced into the rlIA region of the r1589 fusion, also eliminated
rlIB activity. These experiments were important to the use of frameshift
mutations to establish the triplet nature of the genetic code.
Several key concepts underlying the gene fusion approach can be found
in these studies. First, the idea that it is possible to remove a significant
portion of a terminus of a protein (amino terminus in this case) and still
retain sufficient protein function has proved to be the case with a large
number of proteins. Second, the possibility of fusing two different proteins
together and retaining one or both activities was not self-evident. It seemed
quite reasonable to imagine that the generation of a single polypeptide
chain from two chains would result in mutual interference with proper
folding and functioning of each protein. Third, and most importantly, the
notion of using downstream protein activity to report on what was happen-
ing upstream--the reporter gene concept--was key to these studies. This,
of course, is the key feature of the gene fusion approach.
This history has been described as though it was known at the time that
the rlI genes coded for protein. Extraordinarily enough, it was not shown
until many years later that this was the case. Nevertheless, the genetic
evidence was considered compelling enough at the time that the conclusions
of these studies gained widespread acceptance among molecular biologists.
The next steps in the development of gene fusion approaches came
from studies on the lac operon of E. coli. The first fusions of lac were
obtained unwittingly as revertants of strong polar mutations in the lacZ
gene. 4 Selection for restoration of the activity of the downstream lacY gene
yielded many deletions that removed the polar mutation site, the promoter
of lac, and fused the lacy gene to an upstream promoter of an unknown
neighboring gene. In 1965, Jacob and co-workers 5 exploited this approach
to select for fusions in which the lacY gene was put under the control of
an operon involved in purine biosynthesis. This was the first report of a
gene fusion in which the regulation of a reporter gene was determined by
the gene to which it was fused; the Lac permease was regulated by the
concentration of purines in the growth media.
Subsequently, Muller-Hill and Kania 6 showed that the properties of
/3-galactosidase allowed an even broader use of the gene fusion approach
3 F. H. C. Crick, L. Barnett, S. Brenner, and R. J. Watts-Tobin, Nature 192, 1227 (1961).
4 j. R. Beckwith, J. Mol. Biol. 8, 427 (1964).
5 F. Jacob, A. Ullmann, and J. Monod, J. Mol. Biol. 42, 511 (1965).
6 B. Muller-Hill and J. Kania, Nature 249, 561 (1974).
1
THE ALL PURPOSE GENE FUSION 5
in this system. Using a very early chain-terminating mutation, they found
that they could restore/3-galactosidase activity by deleting the polar muta-
tion site and fusing the remaining portion of the polypeptide to the upstream
lacI gene product, the Lac repressor. It was even possible to obtain hybrid
proteins with both repressor and/3-galactosidase activity.
Generalizing the Approach
In all the cases described to this point, genetic fusions were obtained
between two genes that were normally located close to each other on the
bacterial chromosome or on an F' factor. This feature of early gene fusion
studies presented quite strict limitations on the systems that could be ana-
lyzed by this approach. However, beginning first with some old-fashioned
approaches to transposing the lac region to different positions on the chro-
mosome, 7 we began to see that the gene fusion approach might be applied
more widely. A graduate student in the author's laboratory, Malcolm Casa-
daban, then developed improvements on transposition techniques that en-
hanced the ability to fuse lac more generally to bacterial genes. 8 Malcolm
continued these improvements in Stanley Cohen's laboratory at Stanford
University and ultimately in his own laboratory at the University of
Chicago. °1,9
All the approaches described so far involved generation of fusions in
vivo. The arrival of recombinant DNA techniques for cloning and fusing
genes in the mid-1970s provided a tremendous boost to the use of gene
fusions. It became possible to fuse genes from or between any organism
pretty much at will.
Gene Fusions for All Seasons
For many years, the gene fusion tool was considered to be one useful
mainly for studying gene expression and regulation by reporter gene expres-
sion. However, as the ease of generating such fusions grew, other uses
became evident. In 1980, we reported the first case where fusing a reporter
protein to another protein of interest allowed purification of the latter
protein. ~I In this case, fl-galactosidase was fused to a portion of the
cytoplasmic membrane protein, MalF. The unusually large size of
7 j. R. Beckwith, E. R. Signer, and W. Epstein, Cold Spring Harbor Syrup. Quant. BioL 31,
393 (1966).
M. Casadaban, .J Mol. Biol. 104, 541 (1976).
9 M. J. Casadaban and S. N. Cohen, Proc. NatL Acad. Sci. U.S.A. 76, 4530 (1979).
01 M. J. Casadaban and J. Chou, Proc. Natl. Acad. Sci. U.S.A. 81, 535 (1984).
11 H. A. Shuman, T. J. Silhavy, and J. R. Beckwith, .J Biol. Chem. 255, 168 (1980).
11
6 HISTORICAL OVERVIEW
/3-galactosidase allowed ready purification of the hybrid protein, which was
then used to elicit antibody to MalF epitopes, facilitating its purification.
We also showed that gene fusions of/3-galactosidase could be used to
study the signals that determine subcellular protein localization. Fusion of
/3-galactosidase to the MalF protein resulted in membrane localization of
the former protein, 11 and fusion of/3-galactosidase to exported proteins
permitted the genetic analysis of bacterial signal sequences) 3a2
Another important step in the evolution of uses of gene fusions came
with the concept of signal sequence traps. The first development of this
concept came out of the recognition that the bacterial enzyme alkaline
phosphatase is active when it is exported to the periplasm but inactive
when it is retained in the cytoplasm. MT Thus, alkaline phosphatase without
its signal sequence provides an assay for export signals via gene fusion
approaches, i.e., alkaline phosphatase will only be active if one attaches a
region of DNA that encodes a signal sequence, thus reallowing its export.
Hoffman and Wright 51 and Colin Manoil and the author 61 reported sys-
tems-one plasmid, one transposon--that allowed the detection of signal
sequences in random libraries of DNA or in a bacterial chromosome. This
approach has been extended with use of numerous other reporter genes,
including, most prominently, fl-lactamase? 7
Extending beyond the differentiation of exported vs cytosolic proteins,
gene fusion techniques can be evolved to determine subcellular localization
of proteins more generally. Clearly, the use of GFP fusions enhances this
ability. MT In addition, reporter proteins that sense specific features of organ-
elle environment may provide a tool for detecting location and genetically
manipulating signals for the localization process. The report of a GFP that
responds to the pH of its environment may be a harbinger of things to
come) 9 One might imagine GFP derivatives that respond to all sorts of
cellular conditions, e.g., the redox environment.
Finally, gene fusions can be used for the study of protein structure,
protein-protein interactions, and protein folding. The yeast two-hybrid
system described by Fields and Song °2 in 1989 has become a powerful tool
for analyzing aspects of quaternary structure of proteins and for detecting
21 .S .D Emr, .M ,ztrawhcS and .T .J ,yvahliS Proc. Natl. Acad. Sci. U.S.A. ,57 2085 .)8791(
31 p. Bassford dna .J Beckwith, Nature 277, 835 .)9791(
41 .S ,sileahciM H. Inouye, .D Oliver, and .J Beckwith, .J Bacteriol. 154, 663 .)3891(
51 .C Hoffman dna .A Wright, Proc. Natl. Acad. Sci. U.S.A. ,28 7015 .)5891(
61 .C Manoil and .J Beckwith, Proc. Natl. Acad. Sci. U.S.A. ,28 9218 .)5891(
71 y. Zhang dna .J .K Broome-Smith, Mol. Microbiol. ,3 1631 .)9891(
81 D. .S ,ssieW .J .C Chen, .J .M Ghigo, .D Boyd, and .J ,htiwkceB .J BacterioL ,181 805 .)9991(
91 G. ,kc6bneseiM D. .A DeAngelis, dna .J E. Rothman, Nature 394, 291 .)8991(
o2 .S Fields dna .O Song, Nature 340, 542 .)9891(
1 THE ALL PURPOSE GENE FUSION 7
novel protein-protein interactions. Whereas the structure of soluble pro-
teins is accomplished relatively easily by X-ray crystallography techniques,
the structure of membrane proteins still largely resists such approaches.
Gene fusion techniques have been able to contribute to understanding
important features of membrane protein structure. The signal sequence trap
techniques have proved invaluable in the determination of the topological
structure of integral membrane proteins, 12 i.e., fusion of the reporter protein
to intra- or extracytoplasmic domains of membrane proteins usually reports
the location of that domain accurately. Similarly, more recent techniques for
detecting interactions between transmembrane segments of such proteins
should allow the elucidation of additional structural features. 32'22 Although
not so widely employed, gene fusion approaches can aid in the study of
protein folding. Luzzago and Cesareni 42 used a cute fusion approach to
isolate mutants affecting the folding of ferritin. Other such ideas must be
waiting in the wings.
The realm of gene fusions has continually expanded. While this volume
describes a host of different issues that can be studied with this technique,
it seems certain that the expansion will continue.
z2 C. Manoil and J. Beckwith, Science 233, 1403 (1986).
22 j. A. Leeds and J. Beckwith, J. Mol. Biol. ,1182 799 (1998).
32 W. P. Russ and D. M. Engelman, Proc. Natl. Acad. Sci. U.S.A. 96, 863 (1999).
42 A. Luzzago and G. Cesareni, EMBO J. 8, 569 (1989).