Methods of Enzymology. Small GTPases and Their Regulators

418 Pages·1995·7.279 MB·English

by  Balch W.E., Channing J.D., Hall A.

Preface
Rho-related GTP-binding proteins constitute a functionally distinct
group in the small GTPase superfamily. Like Ras, they control intracellular
signal transduction pathways, and it is now firmly established that Rho-
related GTPases regulate the organization of the actin cytoskeleton of all
eukaryotic cells. Accordingly, this family of GTPases controls cell adhesion,
cell movement, and cytokinesis.
This volume describes a wide range of experimental approaches that
have been used to study the function of Rho-related GTPases both in vitro
and in vivo. The availability of recombinant proteins has been of enormous
benefit in characterizing the biochemical and biological activities of the
GTPases and of the proteins with which they interact. The first part of this
volume deals with expression systems used both in Escherichia coli and in
insect cells. The driving force for the enormous interest now being taken
in the Rho family of GTPases stems from their demonstrated biological
roles, particularly as regulators of adhesion and movement. Thus many of
the cellular assays that have been used to establish these effects are included
in this volume. The ultimate test for any cellular activity attributed to a
GTPase is the ability to reconstitute that activity in vitro. To date, this has
been achieved only for Rac-dependent activation of phagocytic NADPH
oxidase, and several chapters are devoted to this topic.
Although the area has already generated an enormous amount of gen-
eral interest, the functional analysis of small GTPases is still in its infancy.
There are many more surprises to come as the biochemical details of
the pathways controlled by small GTPases are elucidated. The prize is a
molecular explanation of many aspects of contemporary cell biology.
We are extremely grateful to all the contributors who have taken the
time to commit their expertise to paper, and are confident that their efforts
will be greatly appreciated by the scientific community. Dr. Hall thanks
the Cancer Research Campaign (UK), the Wellcome Trust, and the Medical
Research Council (UK) for providing the funds and environment that have
allowed him to work in this very exciting area.
ALAN HALL
W. E. BALCH
CHANNING J. DER
iiix
Contributors to Volume 256
Article numbers arc in parentheses following the names of contributors.
Affiliations listed are currenl.
ARIE ABO (5, 29), Onyx ,slacituecamrahP Biochemistry, Emory University Medical
Richmond, California 60849 School, Atlanta, Georgia 22303
PETER ADAMSON (19), Vascular Biology -eR RICHARD A. CERIONE (2, 9, 12), Department
hcraes ,ertneC Kings egelloC London, Lon- of Pharmacology, Cornell ,ytisrevinU Ith-
don, W8 7AH, United Kingdom ,aca New York 35841
DANIEL E. H. AFAR (15), Department of -iM PIERA CICCHETTI (17), Institute for ,sciteneG
ygoloiborc and Molecular ,sciteneG -revinU ytisrevinU of Cologne, Cologne ,47605-D
sity of soL-ainrofilaC Angeles, Los ,selegnA ynamreG
ainrofilaC 42009 DAGMAR DIEKMANN (23), CRC Oncogene
SOHAIL AHMED (14), Department of Neuro- and Signal noitcudsnarT Group, MRC Lab-
,yrtsimehc Institute of Neurology, London oratory for Molecular Cell Biology dna
WC1N 1PJ, United Kingdom, and etutitsnI Department of Biochemistry, University
of Molecular and Cell Biology, National College London, London WC1E 6BT,
ytisrevinU of ,eropagniS Singapore 1150 detinU Kingdom
NOMIS T. DILLON (20), Department of -orciM
KLAUS AKTORIES (21), Institute of Pharma-
biology dna Molecular Biology, Tufts -inU
cology and Toxicology, Albert-Ludwigs
ytisrev School lfq ,enicideM Boston, -assaM
,ytisrevinU D-79104 ,grubierF Germany
sttesuhc 11120
PONTUS ASPENSTROM (25), Department of
OLIVIER DORSEUIL (39), Institut Cochin ed
Zoological Cell Biology, Arrhenius Labo-
euqit4n4G Mol&ulaire, 1NSERM OtinU
seirotar ,5E ehT Wenner-Gren ,etutitsnI
,752 41057 Paris, ecnarF
Stockholm ,ytisrevinU ,19-601S Sweden
ALESSANDRA EVA (38), Laboratory of -ulleC
DAVID BALTIMORE (17), sttesuhcassaM Insti-
ral and raluceloM Biology, National recnaC
tute of Technology, ,egdirbmaC -uhcassaM
Institute, National Institute of Health,
sttes 93120
,adsehteB Maryland 29802
A1C1RTAP BEROEZ-AULLO (32), Laboratoire
LARRY a. FEIG (20), Department of -mehcoiB
ed Biologie Mol(culaire et ,egacneuq4S
,yrtsi Tufts ,ytisrevinU School of ,enicideM
~tisrevinU Bordeaux ,II 33076 Bordeaux,
Boston, sttesuhcassaM 11120
ecnarF
EPPILIHP FORT (18), etutitsnI of raluceloM -eG
JACQUES BERTOGLIO (35), INSERM CJF -39 ,sciten University ,reillepitnoM F 33.043
,10 dtlucaF ed ~tisrevinU-eicamrahP -siraP ,reilleptnoM ecnarF
Sud, 92296 Chatenay Malabry Cedex,
ROSEMARY FOSTER (13), MGM Cancer -neC
ecnarF
ret and Department of ,enicideM Harvard
GARY M. BOKOCH (4, 28), Departments of Medical School, Charlestown, Massachu-
Immunology and Cell Biology, ehT sppircS sttes 92120
Research Institute, La Jolla, California
GERARD GACON (39), Institut Cochin ed -n~G
73029
(ique ,erialuc~loM 1NSERM Unit4 ,752
EC1RTAP BOQUET (32), ~tinU sed Toxines -iM 41057 Paris, ecnarF
,senneiborc Institut Pasteur, 75724 ,siraP MURIELLE GIRY (32), ~tinU sed Toxines -iM
ecnarF ,senneiborc Institut Pasteur, 75724 ,siraP
EDWARD P. BOWMAN (27), Department of. ecnarF
XI
X CONTRIBUTORS TO VOLUME 256
ALAN HALL (1, 8, 23), MRC Laboratory for EDWARD MANSER (16, 24), Institute of -celoM
Molecular Cell Biology and Department of ular and Cell Biology, National ytisrevinU
Biochemistry, University College London, of ,eropagniS Singapore 1150
London WC1E 6BT, England JANET MCCULLOUGH (30), Department of Mi-
CHRISTINE HALL (14), Institute of ,ygolorueN ygoloiborc and Molecular ,sciteneG -revinU
London WC1N 1P ,J United Kingdom sity of Vermont, Burlington, Vermont 50450
JOHN F. HANCOCK (10), Onyx -ituecamrahP TORU MIKI (11), Laboratory of ralulleC and
,slac Richmond, California 60849 Molecular Biology, National Cancer Insti-
MATTHEW J. HART (9), Department of Phar- ,etut National Institutes of ,htlaeH ,adsehteB
,ygolocam Ithaca, New York 35841 Maryland 29802
DOUGLAS I. JOHNSON (30), Department of -iM PETER J. MILLER (30), Department of -orciM
ygoloiborc and Molecular ,sciteneG -revinU biology and Molecular ,sciteneG ytisrevinU
sity of Vermont, Burlington, Vermont05405 of Vermont, Burlington, Vermont 50450
INGO JUST (21), Institute of Pharmacology and TAKAKAZU MIZUNO (3), Department of Mo-
Toxicology, Albert-Ludwigs ,ytisrevinU lecular Biology and Biochemistry, Osaka
40197-D ,grubierF Germany ytisrevinU Medical School, Suita, Osaka
ULLA G. KNAUS (4), Department oflmmunol- ,565 Japan
,yAD The Scripps Research Institute, La
NARITO MORII (22), Department of -amrahP
,alloJ California 73029
,ygoloc Kyoto University Faculty of Medi-
ROBERT KOZMA (14), Institute of ,ygolorueN ,enic Kyoto ,606 Japan
London WC1N IPJ, United Kingdom, and
HIROYUKI NAKANISHI (3), Department of Mo-
Institute of raluceloM and Cell Biology, Na-
ralucel Biology and Biochemistry, Osaka
tional University of Singapore, Singapore
ytisrevinU Medical School, Suita, Osaka
1150
,565 Japan
J. DAVID LAMBETH (27), Department of -DiB
SHUH NARUMIYA (22, 31), Department of
chemistry, Emory University laCideM
,ygolocamrahP Kyoto ytisrevinU Faculty of
School, Atlanta, Georgia 22303
Medicine, Kyoto University, Kyoto ,606
PAUL LANG (35), INSERM CJF ,1O-39 dtlucaF
napaJ
ed ~tisrevinU-eicamrahP Paris-Sud, 69229
yanetahC Malabry Cedex, France MICHAEL F. OLSON (25), CRC Oncogene and
Signal Transduction Group, MRC Labora-
GI~RALD LECA (39), INSERM ,131~tinU
tory for Molecular Cell Biology, ytisrevinU
Association Chlude Bernard, Institute
College London, London WCIE 6BT,
latipOH-eigolotameH'd Saint-Louis, ,siraP
detinU Kingdom
ecnarF
Huort PATERSON (19), Section of Cell and
EMMAUEL LEMICHEZ (32), ~tinU sed Toxines
Molecular Biology, Chester Beatty Labora-
Microbiennes, Institut Pasteur, 75724
,seirot Institute of recnaC ,hcraeseR London
,siraP France
SW3 6B ,J United Kingdom
DAVID LEONARD (2,12), Department of -rahP
macology, Cornell ,ytisrevinU Ithaca, New MARK R. PHILIPS (7), Departments of -ideM
York 35841 cine and Cell Biology, New York ytisrevinU
School of Medicine, New York, New
THOMAS LEUNG (16, 24), Institute of -uceloM
York 61001
ral and Cell Biology, National ytisrevinU of
,eropagniS Singapore 1150 MICHAEL H. PILLINGER (7), Department of
,enicideM New York University School of
Louis LIM (14, 16, 24), Institute of ,ygolorueN
,enicideM New York, New York 61001
London WC1N 1PJ, United Kindgom, and
etutitsnI of raluceloM and Cell Biology, Na- MICHEL R. POPOFF (32), Unit~ sed Toxines
tional University of Singapore, Singapore Microbiennes, Institut Pasteur, 75724
1150 ,siraP ecnarF
CONTRIBUTORS TO VOLUME 256 xi
EMILIO PORFIRI (10), Onyx ,slacituecamrahP pan, and Department of Cell Physiology,
Richmond, California 60849 National Institute for Physiological Sci-
,secne Okagaki ,444 Japan
JAMES POSADA (30), Department of -iborciM
ology and Molecular Genetics, ytisrevinU KENJI TAKAISHI (37), Department of -uceloM
of Vermont, Burlington, Vermont 50450 ral Biology and Biochemistry, Osaka -inU
ytisrev Medical School Suita 565, Japan
MARK T. QUINN (28), Veterinary Molecular
Biology, Department of Microbiology, KAZUMA TANAKA (6), Department of -celoM
Montana State ,ytisrevinU Bozeman, Mon- ular Biology dna Biochemistry, Osaka -inU
tana 71795 ytisrev Medical School Suita, Osaka ,565
napaJ
ANNE J. RIDLEY (33, 3,4), Ludwig Institute
for Cancer ,hcraeseR London WCIP 8BT, TOMOKO TOMINAGA (31), Department of -leC
detinU Kingdom lular dna Molecular Physiology, National
Institute for Physiological Sciences, Oka-
SUSAN E. RITTENHOUSE (26), Jefferson Can-
zaki ,444 Japan
rec Institute and Cardeza Foundation for
DAVID J. UHLINGER (27), Department of
cigolotameH ,hcraeseR ,aihpledalihP Penn-
Biochemistry, Emory University Medical
sylvania 70191
School Atlanta, Georgia 22303
DAVID ROBERTSON (19), Haddow Labora-
A1ME VASQUEZ (39), 1NSERM Unit ,131 As-
,seirot Institute of recnaC ,hcraeseR Sutton,
sociation Claude Bernard Research ,retneC
,yerruS SM2 5NG, United Kingdom
04129 ,tramalC ecnarF
TAKUYA SASAKI (6, 37), Department of Mo-
PIERRE V. VIGNAIS (36), Laboratoire ed Bio-
ralucel Biology and Biochemistry, Osaka
chimie, Departement ed Biologie Molecu-
ytisrevinU Medical School, Suita, Osaka
erial te ,elarutcurtS CEA CEN-Grenoble,
,565 Japan
45083-F Grenoble, ecnarF
ANTHONY W. SEGAL (29), Division of -celoM
SYLVIE VINCENT (18), Institute of raluceloM
ular Medicine, University College London,
,sciteneG University ,reillepitnoM F 33043
London WCIE J6 ,J United Kingdom
,reilleptnoM ecnarF
ANNETTE J. SELF (1, 8), MRC Laboratory for OWEN N. WITrE (15), Molecular Biology In-
Molecular Cell Biology, University egelloC stitute and Howard Hughes Medical Insti-
London, London WC1E 6BT, United ,etut ytisrevinU of California-Los ,selegnA
Kingdom Los Angeles, California 42009
JEFFREY NAMELTTES (13), MGH Cancer -neC DANIELA ILL1RGNAZ (38), Laboratory of -leC
ret and Department of ,enicideM Harvard ralul and Molecular Biology, National -naC
Medical School, Charlestown, -uhcassaM rec Institute, National Institutes of ,htlaeH
setts 92120 ,adsehteB Maryland 29802
MARIE-JOSE STASlA (36), Laboratoire d'En- 1Y ZHENG (2, 9), Department of Pharma-
zymologie, Centre Hospitalier eriatisrevinU cology, Cornell University, Ithaca, New
ed Grenoble, Grenoble, ecnarF York 35841
YOSHIMI TAKAI (3, 6, 37), Department of Mo- MICHAEL ZIMAN (30), Department of -uceloM
lecular Biology dna Biochemistry, Osaka ral and Cell Biology, ytisrevinU of -rofilaC
ytisrevinU Medical School, Osaka ,565 -aJ ,ain Berkeley, Berkeley, California 02749
1 Rho/Rac/G25K MORF E. coli 3
1
Purification of Recombinant Rho / Rac / G25K
from Escherichia coli
By ANNETTE J. SELF and ALAN HALL
Introduction
The purification of Ras-related GTP-binding proteins from recombinant
sources has proved to be invaluable for studying their biochemical proper-
ties and biological effects. The simplest expression systems have made use
of Escherichia coli, although Ras-like GTPases produced in this way are
not posttranslationally modified. Yeast and baculovirus-Sf9 (Spodaptera
frugiperda, full armyworm ovary) insect cells have also been used and since
they are eukaryotic hosts, the GTPases expressed are at least partially
modified. 2'1 A wide range of expression levels has been reported for Ras-
related proteins in E. coli; in the case of Ras, yields of 5.7 mg/liter of
culture have been obtained, 3 whereas others such as Rap1, for example,
have proved much more difficult to make in a stable form. Members of
the Rho family have been relatively difficult to express in E. coli in large
amounts; as described below, we obtain yields of around 0.1-1 rag/liter.
The mammalian Rho subfamily consists of RhoA, B, and C, Racl and
2, G25K/CDC42, RhoG, and TC10. 9-4 These proteins are %03 identical to
Ras in amino acid sequence and %55 identical to each other, and their
overall three-dimensional structure is expected to be very similar to that
of Ras. °1 RhoA, B, and C are %58 identical to each other, with almost all
x .S G. Clark, J. P. McGrath, and A. D. Levinson, Mol. Cell Biol. 5, 2726 (1985).
2 M. J. Page, A. Hall, .S Rhodes, R. H. Skinner, V. Murphy, M. Sydenham, and P. N. Lowe,
.J Biol. Chem. 264, 19147 (1989).
3 A. M. De Vos, L. Tong, M. V. Milburn, P. M. Matias, J. Jancarik, .S Noguchi, S. Nishimura,
K. Mitra, E. Ohtsuka, and S. Kim, Science 239, 888 (1988).
4 p. Madaule and R. Axel, Cell 41, 31 (1985).
5 j. Didsbury, R. F. Weber, G. M. Bocock, T. Evans, and R. Synderman, J. Biol. Chem. 264,
16378 (1989).
6 K. Shinjo, J. G. Koland, M. J. Hart, V. Naraismham, D. J. Johnson, T. Evans, and R. A.
Cerione, Proc. Natl. Acad. Sci. U.S.A. 87, 9853 (1990).
7 .S Munemitsu, M. A. Innis, R. Clark, F. McCormick, A. Ullrich, and P. Polakis, Mol. Cell.
Biol. 10, 5977 (1990).
s G. T. Drivas, A. Shih, E. Coutavas, M. G. Rush, and P. D' Eustachio, Mol. Cell. Biol. 10,
1793 (1990).
9 S. Vincent, P. Jeanteur, and P. Fort, MoL Cell Biol. ,21 3138 (1992).
01 E. F. Pai, W. Kabsch, U. Krengal, K. C. Holmes, J. John, and A. Wittinghofer, Nature 341,
209 (1989).
Copyright © 1995 by Academic Press, Inc.
METHODS IN ENZYMOLOGY, VOL. 256 All rights of reproduction in any form reserved.
4 NOISSERPXE DNA NOITACIFIRUP l
of the divergence being at the carboxy-terminal end of the proteins; Racl
and 2 are 92% identical to each other with 15 amino acids different; and
G25K and CDC42Hs are the closest related isoforms with only 9 amino
acid differences between them. All Rho family members contain a C-
terminal CAAX box motif (A = aliphatic amino acid; X = L for Rho and
Rac; X = F for CDC42/G25K), and all are posttranslationally modified in
vivo by the addition of a C 20 geranylgeranyl isoprenoid, u 31 Interestingly,
RhoB also appears to be a substrate for the farnesyltransferaseJ 4
Like all small GTPases, the Rho-related proteins are regulated by gua-
nine nucleotide exchange factors (GEFs) and GTPase-activating proteins
(GAPs), and the characterization of these regulatory proteins has relied
on a source of recombinant protein. All GAPs and most GEFs are active
in vitro on E. coli-produced, nonmodified Rho-related GTPases. E. coli-
produced recombinant proteins are also very useful for studying the biologi-
cal function of the Rho subfamily by microinjection because the GTPases
become posttranslationally modified and functionally active after in-
jectionJ 5
To characterize the function of Rho-related proteins, we have purified
RhoA, Racl, and G25K from E. coli using the glutathione S-transferase
(GST) gene fusion vector pGEX-2T (Pharmacia LKB Biotechnology,
Inc.). 61 As described in the following section, the yields of these proteins
from this vector are not as high as have been reported for other proteins
expressed using this system, but purification is extremely rapid and the
final preparations are of high purity.
Construction of Vectors
cDNAs generated by the polymerase chain reaction (PCR) and encod-
ing human RhoA, Racl, and G25K were fused to the carboxy-terminal
end of the Schistosoma aponicum glutathione S-transferase gene by cloning
into the BamHI/EcoRI sites of pGEX-2T (see Fig. 1). Expression of the
fusion protein is under the control of the tac promoter, and the nucleotide
sequences across the fusion junctions are shown in Fig. lb. After cleavage
.M11 Katayama, .M Kawata, .Y Yoshida, H. Horiuchi, .T Yamamoto, .Y Matsuura, and
.Y Takai, .J Cell. Biol. 266, 93621 .)1991(
21 .B .T Kinsella, R. A. Erdman, and .W A. Maltese, .J Biol. Chem. ,51 6879 .)1991(
31 H. Yamane, .C .C Farnsworth, H. Xiec, .T Evans, .W .N Howald, .M H. Gelb, J. A. Glomset,
.S Clarke, and .B .K .K Fung, Proc. Natl. Acad. Sci. U.S.A. ,88 682 .)1991(
4a p. Adamson, .C J. Marshall, A. Hall, and .P A. Tilbrook, .J Biol. Chem. ,762 33002 .)2991(
.H51 .F Paterson, A. J. Self, .M D. Garrett, I. Just, .K Aktories, and A. Hall,
.J Cell Biol. 111, 1001 .)0991(
61 D. .B Smith and .K .S Johnson, Gene ,76 13 .)8891(
1 Rho/Rac/G25K MORF E. coli 5
b THROMBIN
ueLI
Val orP Arg~Gly serlpro yIG lie siH Arg Asp
TSG .......... CTG F-I-G GCC TGC AGG CCT GCC AGG ATT CAT CGT GAC TGA GTC GCA
I I I I
BamHl __ EcoRl Stop codons
lamS
TSG .......... GTC G-I-r GCC TGC AGG CCT GCC GCT....rhoA
TSG .......... GTC GTT GCC TGC AGG CCT GCC CAG.,..racl
TSG .......... GTC GTT GCC TGC SGG CCT GCC CAG.,..GZSK
codon
2
FIG. .1 Structure of the glutathione S-transferase vector pGEX-2T. (a) Schematic represen-
tation of pGEX-2T. (b) Nucleotide sequence of pGEX-2T and of pGEX-2T containing RhoA,
Racl, dna G25K cDNAs across the fusion junction.
with thrombin it is predicted that the GTPases will each have Gly-Ser-Pro
fused to the second codon of the native sequence. The pGEX-2T vectors
containing RhoA, Racl, and G25K were each introduced into the E. coli
strain JM101 and stored as glycerol stocks at -70 .°
Purification of Wild-Type RhoA, Rac 1, and G25K
Growth and Purification
One hundred milliliters of L-broth containing 50/~g/ml ampicillin is
inoculated with E. coli containing the expression plasmids taken from the
6 EXPRESSION AND PURIFICATION l
glycerol stock. After overnight incubation at 73 ,° the culture is diluted 1 : 01
into fresh, prewarmed (37 )° L-broth/ampicillin and is incubated for 1 hr in
two 2-liter flasks in a bacterial shaker at 37 .° To induce fusion protein
expression, isopropyl-/3-D-thiogalactopyranoside (IPTG) is added to 1.0
mM (0.5 ml of a 1.0 M stock made in water and stored at -20°), and the
culture is incubated with shaking for a further 3 hr. After induction, the
cells are collected in l-liter buckets by centrifugation at 4000 rpm for 01
min at 4 ° and then resuspended (on ice) in 3 ml of cold lysis buffer 05
mM Tris-HC1, pH 7.6, 50 mM NaC1, 5 mM ,21CgM 1 mM dithiothreitol
(DTT), and 1 mM phenylmethylsulfonyl fluoride (PMSF).
We have noted that many purification procedures for GST fusion pro-
teins use buffers containing phosphate, a chelator of magnesium ions. 61 In
low magnesium concentrations, Rho-related GTPases rapidly lose their
bound guanine nucleotide (see 9 in this volume) and are unstable. It is
therefore important that phosphate buffers or other chelators of magnesium
such as EDTA are not used in the purification procedure and that there
is an excess of free magnesium in all buffers used.
Resuspended bacteria are lysed by sonication on ice (three times at 1
min each). We use a small probe on an MSE Soniprep 051 sonicator at an
amplitude of 41 tzm, and the bacterial suspension is kept cool at all times.
As lysis occurs the suspension turns from a light creamy color to a muddy
brown and becomes somewhat more viscous. The sonicate is centrifuged
at 10,000 rpm for 01 min at 4 ,° and the supernatant (4 ml) is carefully
transferred to a 5-ml bijou tube (Sterillin). Some 30-50% of GST-RhoA,
GST-Racl, and GST-G25K produced by this expression system in JM101
is found in the pellet after centrifugation of the sonicate.
Glutathione-agarose beads (Sigma G4510) or glutathione-Sepharose
4B beads (Pharmacia) are prewashed with several volumes of lysis buffer
and kept as a 1 : 1 suspension. One milliliter of this suspension is added to
the supernatant and is incubated for 30 min on a rotating wheel at 4 .° The
beads are pelleted in a benchtop centrifuge at 4000 rpm for 1 min, and the
supernatant is removed and discarded. The beads are then washed with 5
ml of cold lysis buffer (without DTT and PMSF) five times to remove
unbound proteins. Recovery of bound protein can be achieved in one of
two ways.
.a Recovery of Fusion Protein. The GST fusion protein can be eluted
from the beads by competition with free glutathione. An equal volume (0.5
ml) of freshly prepared release buffer 05 mM Tris-HC1, pH 8.0, 051 mM
,1CaN 5 mM ,21CgM 1 mM DTT + 5 mM reduced glutathione (Sigma
G4251) (final pH 7.5), is added to the washed beads and incubated for 2
min at 4 ° on a rotating wheel. The beads are pelleted and the supernatant
11 Rho/Rac/G25K MORF E. coli 7
is removed. The procedure is repeated, and the two supernatants are pooled
(1 ml) and dialyzed overnight (see later).
.b Recovery of Nonfused Rho/Rac/G25K. The washed beads (0.5 ml)
are transferred to a 1.5-ml microcentrifuge tube and resuspended in 0.5 ml
of thrombin digestion buffer (50 mM Tris-HC1, pH 8.0, 150 mM NaC1, 2.5
mM CaCI2,5 mM MgCI2, 1 mM DTT) containing 5 units of bovine thrombin
(Sigma T6634). The suspension is incubated at 4 ° on a rotating wheel
overnight. After thrombin digestion, the beads are pelleted in a microcentri-
fuge (1 min), and the supernatant is removed. Sometimes after thrombin
digestion, the cleaved protein remains partly associated with the beads so
we routinely incubate the beads with another 0.5 ml of high salt/DTY buffer
(50 mM Tris-HC1, 7.6, 150 mM NaC1, 5 mM MgC12, 1 mM DTT) for 2
rain at 4 .° After centrifugation the two supernatants are pooled (1 ml). The
efficiency of thrombin cleavage of GST-RhoA and GST-Racl approaches
100%, but GST-G25K is more resistant and usually only 50% is cleaved
by an overnight incubation with thrombin.
Thrombin can be removed by adding 10 1~ of a suspension of p-amino-
benzamidine-agarose beads (Sigma) to the supernatant and incubating for
a further 30 rain at 4 ° on a rotating wheel.
Dialysis and Storage
For microinjection purposes we dialyze against 2 liters of 10 mM Tris-
HC1, pH 7.6, 150 mM NaC1, 2 mM MgC12, and 0.1 mM DTT at 4 ° overnight
with one buffer change. For GTPase assays where a low salt concentration
is required (10 mM NaC1), we dialyze against 10 mM Tris-HCl, pH 7.6, 2
mM MgC12, and 0.1 mM DTT. Proteins are concentrated to approximately
150/xl in an Amicon Centricon 10 filter device by centrifugation in a fixed
angle rotor at 7000 rpm. We routinely store the final protein preparations
at approximately 1 mg/ml in 10-/zl aliquots, snap frozen in liquid nitrogen.
The protein concentration is determined by a 3HGTP/3HGDP binding
assay as described below. The yield of wild-type proteins as determined by
nucleotide binding is in the order of 0.1-0.2 rag/liter of bacterial culture.
Figure 2 shows a Coomassie-stained gel of GST fusion and thrombin-
cleaved RhoA, N25RhoA (see later), Racl, and G25K proteins.
Determination of Protein Concentration
Protein concentration is determined by a guanine nucleotide nitrocellu-
lose filter binding assay. We use 3HGTP or 3HGDP but 32p-labeled
nucleotides can also be used. Samples of concentrated protein (0.1, 0.2,
8 EXPRESSION AND PURIFICATION [ 1]
1 2 3 4 5 6 7 8 9 kD
t '~1,-- 69
Plllmq qlll, tlllBP ~ ~I--46
~_..
~ ~ ,~,..-3o
g~21.5
FI6. 2. Purification of fusion and thrombin-cleaved proteins. Samples loaded are GST
(lane 1), GST-wild-type RhoA (lane 2), GST-N25RhoA (lane 3), GST-Racl (lane 4), GST-
G25K (lane 5), wild-type RhoA (lane 6), N25RhoA (lane 7), Racl (lane 8), and G25K (lane 9).
and 0.3/zl) are incubated in a total volume of 40/zl of assay buffer (50
mM Tris-HC1, pH 7.6, 50 mM NaC1, 5 mM MgC12, 5 mM DT]?) containing
10 mM EDTA and 0.5/zl [3H]GTP or [3H]GDP (Amersham, 10 Ci/mmol,
1 mCi/ml) for 10 min at 30 .° Samples are diluted with 1 ml of cold assay
buffer (without DTT) and are filtered through prewetted 25-ram nitrocellu-
lose filters (NC45 Schleicher & Schuell 0.45/zm) using a Millipore filtration
device. The filters are washed three times with 3 ml of cold assay buffer
(without DTT) and are allowed to dry in air. Radioactivity is determined
by scintillation counting. If 1 tool of Rho binds 1 mol of [3H]GTP, then 1
/zg Rho should yield 10 6 dpm (disintegrations per minute). The concentra-
tion of the protein sample (mg/ml) is calculated using Eq. (1):
[Protein] cpm//zl 100
= 601 x counting efficiency" (1)
In our hands counting efficiency can be as low as 20%.
Protein concentration can also be determined by comparing samples
with bovine serum albumin (BSA) standards after electrophoresis on a
12% polyacrylamide gel and staining with Coomassie Brilliant Blue R
(Sigma). The concentration of Rho proteins determined by this method is
3- to 5-fold higher than that determined by guanine nucleotide binding.
The estimation of protein concentration by Bradford or Lowry methods
gives values approximately 10-fold higher than those determined by guanine
nucleotide binding. We do not understand the reason for the differences
in the three assays, but a similar discrepancy has been found by others and
also with Ras protein preparations. We use the guanine nucleotide binding
assay as a measure of protein concentration.
Protein Stability
We previously reported that wild-type RhoA produced as a nonfusion
protein in a trp promoter expression system was biologically inactive after