Small GTPases and Their Regulators Part A

543 Pages·1995·10.16 MB·English

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

Preface
The frequent association of mutated Ras proteins with human cancers
has stimulated considerable interest in the role of these small GTPases. A
continuing expansion of interest in Ras family proteins has prompted the
compilation of the chapters in this volume which cover four broad experi-
mental approaches for studying Ras biochemistry and biology. The first
section describes methods for purifying recombinant Ras proteins and the
analysis of their posttranslational modifications. In particular, two chapters
describe the use of farnesyltransferase inhibitors to study Ras function in
vivo. The second section describes in vitro and in vivo approaches to evalu-
ate the guanine nucleotide binding properties of Ras proteins. The third
section emphasizes approaches to measure protein-protein interactions
between components of the Ras signal transduction pathway. The final
section describes diverse protocols for evaluating the biological properties
of Ras proteins.
It is now evident that Ras proteins are members of a large superfamily
of small GTPases. These Ras-related proteins function in diverse cellular
processes such as growth control (Ras family proteins), actin cytoskeletal
organization (Rho family proteins), and intracellular transport (Rab, ARF,
Sarl, and Ran family proteins). Because of the rapid expansion of interest
in these new areas of study, Rho and transport GTPases are covered in
depth in two companion volumes of Methods in Enzymology, 256 and 257.
Techniques applicable to one family are frequently useful for studying other
families. This three-volume series provides a comprehensive collection of
techniques that will greatly benefit research in the field of small GTPase
function, providing both an experimental reference for the many scientists
who are now working in the field and a starting point for newcomers who
are likely to be enticed into it in the years to come.
We are very grateful to all the authors for their time and expertise in
compiling this collection of experimental protocols. These volumes should
provide a resource for addressing the role of members of the Ras superfam-
ily in the biology of normal and transformed cells.
CHANNINGJ . DER
W.E. BALCH
ALANHALL
Contributors to Volume 255
Article numbers are.i n parentheses following the names of contributors.
Affiliations listed are current.
NILS B. ADEY (50) Department of Biology, tory for Physiological Chemistry, University
University of North Carolina at Chapel Hill, of Utrecht, Utrecht, The Netherlands
Chapel Hill, North Carolina 27599 HONG CAI (23) Dana-Farber Cancer Institute
DARIO R. ALESSI (29), MRC Protein Phos- and Department of Pathology, Harvard
phorylation Unit, Department of Biochem- Medical School, Boston, Massachusetts
istry, University of Dundee, Dundee DDI 02115
4HN, Scotland SHARON L. CAMPBELL-BURK (l), Department
ALAN ASHWORTH (29) Chester Beatty Labo- of Biochemistry and Biophysics, University
ratories, Institute of Cancer Research, Lon- of North Carolina at Chapel Hill, Chapel
don SW3 6JB, United Kingdom Hill, North Carolina 27599
JOSEPH AVRUCH (33), Diabetes Unit and Med- JOHN W. CARPENTER (l), Department of Bio-
ical Services, Department of Medicine, chemistry and Biophysics, University of
Harvard Medical School, Massachusetts North Carolina at Chapel Hill, Chapel Hill,
General Hospital East, Cambridge, Massa- North Carolina 27599
chusens 02129 DAVID CASTLE (27), Department of Cell Biol-
ogy and Anatomy, University of Virginia
DAFNA BAR-SAGI (13,43), Cold Spring Har-
Health Sciences Center, Charlottesville, Vir-
bor Laboratory, Cold Spring Harbor, New
ginia 22908
York II 724
ANDREW D. CATLING (25), Department of Mi-
RHONDA L. BOCK (38) Department of Cancer
crobiology and Cancer Center, School of
Research, Merck Research Laboratories,
Medicine, University of Virginia, Char-
West Point, Pennsylvania 19486
lottesville, Virginia 22908
GIDEON E. BOLLAG (2,3,18), Onyx Pharma-
RITA S. CHA (44), Center for Environmental
ceuticals, Richmond, California 94806
Health Sciences, Massachusetts Institute of
JOHANNES L. Bos (17, 22) Laboratory for Technology, Cambridge, Massachusetts
Physiological Chemistry, University of 02139
Utrecht, Utrecht, The Netherlands
PIERRE CHARDIN (13), Institute de Pharma-
DAVID A. BRENNER (35), Departments of cologie Moleculaire et Cellulaire, 06560 Val-
Medicine, Biochemistry and Biophysics, bonne, France
University of North Carolina at Chapel Hill, LI CHEN (46), Onyx Pharmaceuticals, Rich-
Chapel Hill, North Carolina 27599 mond, California 94806
DANIEL BROEK (15), Department of Biochem- ROBIN CLARK (2), Onyx Pharmaceuticals,
istry and Molecular Biology, Norris Com- Richmond, California 94806
prehensive Cancer Center, University of
GEOFFREY J. CLARK (40), Department of
Southern California School of Medicine,
Pharmacology, School of Medicine, Univer-
Los Angeles, California 90033
sity of North Carolina at Chapel Hill,
MICHAEL S. BROWN (5), Department of Mo- Chapel Hill, North Carolina 27599
lecular Genetics, University of Texas South- PHILIP COHEN (29) MRC Protein Phosphory-
western Medical Center, Dallas, Texas lation Unit, Department of Biochemistry,
75235 University of Dundee, Dundee DDI 4HN
BOUDEWUN M. T. BURGERING (22) Labora- Scotland
ix
X CONTRIBUTORS TO VOLUME 255
ROBBERT H. COOL (lo), Max-Planck-Institut ROBERT FINNEY (32), Molecular Cancer Biol-
fiir Molekulare Physiologie, 44139 Dort- ogy, Cell Therapeutics, Seattle, Washing-
mund, Germany ton 98119
GEOFFREY M. COOPER (23), Dana-Farber MA~HIAS FRECH (13) Institute de Pharma-
Cancer Institute and Department of Pathol- cologie Moleculaire et Cellulaire, 06560 Val-
ogy, Harvard Medical School, Boston, Mas- bonne, France
sachusetts 02115 JACKSON B. GIBBS (12, 19, 38), Department
SALLY COWLEY (29), Chester Beatty Labora- of Cancer Research, Merck Research Labo-
tories, Institute of Cancer Research, London ratories, West Point, Pennsylvania 19486
SW3 6JB, United Kingdom JOSEPH L. GOLDSTEIN (5) Department of Mo-
ADRIENNE D. Cox (21, 40), Departments of lecular Genetics, University of Texas South-
Radiation Oncology and Pharmacology, western Medical Center, Dallas, Texas
School of Medicine, University of North 75235
Carolina at Chapel Hill, Chapel Hill, North SUZANNE M. GRAHAM (40), Department of
Carolina 27599 Pharmacology, School of Medicine, Univer-
DIDIER CUSSAC( 13), Institutede Pharmacolo- sity of North Carolina at Chapel Hil, Chapel
gie Moleculaire et Cellulaire, 06560 Val- Hill, North Carolina 27599
bonne, France HIDESABURO HANAFUSA (37) Laboratory of
ALIDA M. M. DE VRIES-SMITS (17,22), Labo- Molecular Oncology, Rockefeller Univer-
ratory for Physiological Chemistry, Univer- sity, New York, New York 10021
sity of Utrecht, Utrecht, The Netherlands JOHN F. HANCOCK (2,7,24), Onyx Pharma-
PAUL DENT (27) Howard Hughes Medical ceuticals, Richmond, California 94806
Institute, and Markey Center for Signal MATT J. HART (14), Onyx Pharmaceuticals,
Transduction, University of Virginia Health Richmond, California 94806
Sciences Center, Charlottesville, Virginia CRAIG A. HAUSER (41), Cancer Research
22908 Center, La Jolla Cancer Research Founda-
CHANNING J. DER (6,21,40), Department of tion, La Jolla, California 92037
Pharmacology, The University of North DESIREE HERRERA (32), Molecular Cancer
Carolina at Chapel Hill, Chapel Hill, North Biology, Cell Therapeutics, Seattle, Wash-
Carolina 27599 ington 98119
JULIAN DOWNWARD (11,17), Imperial Cancer STANLEY M. HOLLENBERG (34) Vellum Insti-
Research Fund, London, WC2A 3PX, tute, Portland, Oregon 97201
United Kingdom GUY L. JAMES (5) Department of Molecular
CHRISTINE ELLIS (20), Institute of Cancer Re- Genetics, University of Texas Southwestern
search, Chester Beatty Laboratories, Lon- Medical Center, Dallas, Texas 75235
don SW3 6JB, United Kingdom MICHEL JANICOT (42), Rhone-Poulenc Rorer,
TONY EVANS (2) Onyx Pharmaceuticals, Centre de Recherche de Vitry/Alfortville,
Richmond, California 94806 94403 Vitry sur Seine, France
STEPHAN M. FELLER (37), Laboratory of Mo- ALGIRDAS J. JESAITIS (48) Department of Mi-
lecular Oncology, Rockefeller University, crobiology, Montana State University,
New York, New York 10021 Bozeman, Montana 59717
JEFFREY FIELD (47) Department of Pharma- WEI JIANG (45) Molecular Biology and Virol-
cology, University of Pennsylvania School ogy Laboratory, The Salk Institute, La
of Medicine, Philadelphia, Pennsylvania Jolla, California 92037
I9104 GARY L. JOHNSON (30) Division of Basic Sci-
CATHY FINLAY (39), Department of Cell Biol- ences, National Jewish Center for Immunol-
ogy, Glaxo Inc., Research Triangle Park, ogy and Respiratory Medicine, Denver,
North Carolina 27709 Colorado 80206, and Department of Phar-
CONTRIBUTORS TO VOLUME 255 xi
macology, University of Colorado Medical BEN MARGOLIS (36), Department of Pharma-
School, Denver, Colorado 80262 cology, and Kaplan Cancer Center, New
York University Medical Center, New York,
J. DEDRICK JORDAN (21), Department of
New York 10016
Chemistry, School of Medicine, University
of North Carolina at Chapel Hill, Chapel CHRISTOPHERJ . MARSHALL (28, 29), Chester
Hill, North Carolina 27599 Beatty Laboratories, Institute of Cancer Re-
search, London SW3 6JB, United Kingdom
Scold M. KAHN (45), Center for Radiological
Research, Columbia University, New York, MARK S. MARSHALL (33) Department of
New York 10032 Medicine, Division of Hematology and On-
cology, and Walther Oncology Center, Indi-
BRIAN K. KAY (50) Curriculum in Genetics
ana University, Indianapolis, Indiana 46202
and Department of Biology, University of
FRANK MCCORMICK (3, 18), Onyx Pharma-
North Carolina at Chapel Hill, Chapel Hill,
ceuticals, Richmond, California 94806
North Carolina 27599
VIVIEN MEASDAY (20), Banting and Best De-
YOSHITO KAZIRO (16) Faculty of Bioscience
partment of Medical Research, University
and Biotechnology, Tokyo Institute of
of Toronto, Toronto, Canada M5G IL6
Technology, Yokohama 226, Japan
ANDREI MIKHEEV (44), Center for Environ-
MIREI~LE KENIGSBERG( 42), Rhone-Poulenc
mental Health Sciences, Massachusetts Insti-
Rorer, Centre de Recherche de Vitry/Alfort-
tute of Technology, Cambridge, Massachu-
ville, 94403 Vitry sur Seine, France
setts 02139
ROYA KHOSRAVI-FAR (6) Department of
KEITH A. MINTZER (47), Department of Phar-
Pharmacology, School of Medicine, Univer-
macology, University of Pennsylvania
sity of North Carolina at Chapel Hill,
School of Medicine, Philadelphia, Pennsyl-
Chapel Hill, North Carolina 27599
vania 19104
BEATRICE KNUDSEN (37), Laboratory of Mo-
HIROSHI MITSUZAWA (9), Department of Mi-
lecular Oncology, Rockefeller University,
crobiology and Molecular Genetics, Univer-
New York, New York 10021
sity of California at Los Angeles, Los
NANCY E. KOHL (38) Department of Cancer Angeles, California 90024
Research, Merck Research Laboratories, MICHAEL F. MORAN (20), Banting and Best
West Point, Pennsylvania 19486 Department of Medical Research, Univer-
SHINYA KURODA (26) Department of Molec- sity of Toronto, Toronto, Canada MSG I L6
ular Biology and Biochemistry, Osaka Uni- DEBORAH K. MORRISON (31), Cellular
versity Medical School, Okazaki 444, Ja- Growth Mechanisms Group, ABL-Basic
pan, and Department of Cell Physiology, Research Program, NCI-FCRDC, Freder-
National Institute for Physiological Sci- ick, Maryland 21702
ences, Okazaki 444, Japan SCOTTD . MOSSER( 38) Department of Cancer
CAROL A. LANGE-CARTER (30) Division of Research, Merck Research Laboratories,
Basic Sciences, National Jewish Center for West Point, Pennsylvania 19486
Immunology and Respiratory Medicine, RAYMOND D. MOSTELLER (15), Department
Denver, Colorado 80206, and Department of Biochemistry and Molecular Biology,
of Pharmacology, University of Colorado Norris Comprehensive Cancer Center, Uni-
Medical School, Denver, Colorado 80262 versity of Southern California School of
SALLY J. LEEVERS (28, 29), Chester Beatty Medicine, Los Angeles, California 90033
Laboratories, Institute of Cancer Research, ALLEN OLIFF (38) Department of Cancer Re-
London SW3 6JB, United Kingdom search, Merck Research Laboratories, West
CHRISTIAN LENZEN (lo), Max-Planck-Insti- Point, Pennsylvania, 19486
tute fur Molekulare Physiologie, 44139 WEONMEE PARK (15), Department of Biologi-
Dortmund, Germany cal Sciences, Molecular Biology Program,
xii CONTRIBUTORS TO VOLUME 255
University of Southern California, Los National Institute for Physiological Sci-
Angeles, California 90089 ences, Okazaki 444, Japan
CHARLESA . PARKOS( 48), Department of Pa- EDWARD Y. SKOLNIK (36) Departments of
thology, Brigham and Women’s Hospital, Pharmacology and Internal Medicine, Skir-
Boston, Massachusetts 02115 ball Institute for Biomolecular Medicine,
New York University Medical Center, New
MANUEL PEIwCHO (45), California Institute
York, New York 10016
of Biological Research, La Jolla, Califor-
nia 92037 PATRICIA A. SOLSKI (21), Department of
Pharmacology, School of Medicine, Univer-
PAUL POLAKIS (A), GnyX Pharmaceuticals,
sity of North Carolina at Chapel Hill,
Richmond, California 94806
Chapel Hill, North Carolina 27599
EMILIO PORFIRI (2), Onyx Pharmaceuticals,
ANDREW B. SPARKS( 50) Curriculum in Ge-
Richmond, California 94806
netics and Molecular Biology, University of
PATRICK POULLET (49), Department of Micro- North Carolina at Chapel Hill, Chapel Hill,
biology and Molecular Genetics, University North Carolina 27599
of California at Los Angeles, Los Angeles, JEFFRYB . STOCK( 8), Departments of Molecu-
California 90024 lar Biology and Chemistry, Lewis Thomas
Scan POWERS( 14, 46) Onyx Pharmaceuti- Laboratory, Princeton University,
cals, Richmond, California 94806 Princeton, New Jersey 08544
LAWRENCEA . QUILLIAM (41,50), Department THOMAS W. STIJRGILL (27) Howard Hughes
of Pharmacology, University of North Car- Medical Institute, and Markey Center for
olina at Chapel Hill, Chapel Hill, North Signal Transduction, University of Virginia
Carolina 27599 Health Sciences Center, Charlottesville, Vir-
MARK T. QUINN (48) Veterinary Molecular ginia 22908
Biology, Montana State University, Boze- YOSHIMI TAKAI (26) Department of Molecu-
man, Montana 59717 lar Biology and Biochemistry, Medical
School, Osaka University, Osaka 565,
CHRISTOPHW . M. REUTER (25), Department
Japan
of Microbiology and Cancer Center, School
FUYUHIKO TAMANOI (9, 49) Department of
of Medicine, University of Virginia, Char-
Microbiology and Molecular Genetics, Uni-
lottesville, Virginia 22908
versity of California at Los Angeles, Los
GUILLERMO ROMERO (27) Department of
Angeles, California 90024
Pharmacology, University of Pittsburgh,
TRAC( J. THOMAS (38) Department of Cancer
Pittsburgh, Pennsylvania 15261
Research, Merck Research Laboratories,
BONNEE RUBINFELD (4), Onyx Pharmaceuti-
West Point, Pennsylvania 19486
cals, Richmond, California 94806
JUDITH M. THORN (50) Department of Biol-
TAKAYA SATOH (16), Faculty of Bioscience ogy, University of North Carolina at Chapel
and Biotechnology, Tokyo Institute of Hill, Chapel Hill, North Carolina 27599
Technology, Yokohama 226, Japan
BRUNO TOCQUE (42), Rhone-Poulenc Rorer,
MICHAEL D. SCHABER (19) Department of Centre de Recherche de Vitry/Alfortville,
Cancer Research, Merck Research Labora- 94403 Vitry sur Seine, France
tories, West Point, Pennsylvania 19486 LOESJEV ANDERVOORN (17),Laboratory for
JOSEPH SCHLESSINGER( 36) Department of Physiological Chemistry, University of
Pharmacology, New York University, Med- Utrecht, Utrecht, The Netherlands
ical Center, New York, New York 10016 ANNE B. VOITEK (34) Fred Hutchinson Can-
KAZUVA SHIMIZU (26) Department of Molec- cer Research Center, Seattle, Washington
ular Biology and Biochemistry, Osaka Uni- 98104
versity Medical School, Okazaki 444, Ja- CRAIG VOLKER (8), Departments of Molecu-
pan, and Department of Cell Physiology, lar Biology and Chemistry, Lewis Thomas
. . .
CONTRIBUTORS TO VOLUME 255 x111
Laboratory, Princeton University, TAI W~I WONG (37) Department of Bio-
Princeton, New Jersey 08544 chemistry, University of Medicine and Den-
MICHAEL J. WEBER (25), Department of Mi- tistry of New Jersey (UMDNJ), Piscataway,
crobiology and Cancer Center, School of New Jersey 08854
Medicine, University of Virginia, Char- BUNPEI YAMAMORI (26) Department of Mo-
lottesville, Virginia 22908 lecular Biology and Biochemistry, Osaka
I. BERNARD WEINSTEIN (45), Columbia Pres- University Medical School, Okazaki 444,
byterian Cancer Center, New York, New Japan, and Department of Cell Physiology,
York 10032 National Institute for Physiological Sci-
JOHN K. WESTWICK (35, 41), Department of ences, Okazaki 444, Japan
Pharmacology, University of North Caro- HELMUT ZARBL (44) Fred Hutchinson Can-
lina at Chapel Hill, Chapel Hill, North Car- cer Research Center, Seattle, Washington
olina 27599 98104, and Massachusetts Institute of Tech-
FRANCINE R. WILSON (38) Department of nology, Cambridge, Massachusetts 02139
Cancer Research, Merck Research Labora- XIAN-FENG ZHANG (33), Diabetes Unit and
tories, West Point, Pennsylvania 19486 Medical Services, Department of Medicine,
ALFRED WITTINGHOFER (lo), Max-Planck- Harvard Medical School, Massachusetts
Institut ftir Molekulare Physiologie, 44139 General Hospital, Charlestown, Massachu-
Dortmund, Germany setts 02129
Ras
i REFOLDING AND PURIFICATION OF PROTEINS 3
1 Refolding and Purification of Ras Proteins
By SHARON L. CAMI'BELL-BURK and JOHN W. CARPENTER
Introduction
Ras proteins are essential components of cellular processes, providing
a link between growth factor receptors at the cell surface and gene expres-
sion in the nucleus to regulate normal cell growth and differentiation. -~'~
They are often referred to as "molecular switches" because they regulate
intracellular signaling by a cyclic process involving interconversion between
GTP (on) and GDP (off) states. The ras gene product, p21, has become
an essential reagent in many laboratories interested in Ras-mediated sig-
nal transduction.
Our laboratory has been investigating the structural basis for Ras func-
tion using nuclear magnetic resonance (NMR) spectroscopy. These studies
require tens of milligrams of isotopically 15N,13C-enriched material, and
therefore efforts have been made to increase the yield and reduce the
cost associated with isolation of isotopically enriched Ras by optimizing
purification methods. When H-Ras si produced using the expression system
of Feig et al., 3 95-99% si localized in the inclusion bodies as insoluble
protein, whereas 1-5% si expressed in the soluble fraction. Consequently,
we have worked out a procedure for refolding Ras proteins from inclu-
sion bodies, to optimize the overall yield of Ras protein isolated from
Escherichia coll. Here we describe purification methods for isolating
Ras proteins in high yield from both soluble and particulate fractions of
E. coll. Ras protein refolded from inclusion bodies possesses biochemical
activities comparable to Ras protein purified from the soluble fraction.
Furthermore, NMR data indicate that the refolded Ras protein si structur-
ally similar to Ras isolated from the soluble fraction. The purification
procedures should be applicable to a number of low molecular weight
Ras-related proteins that share sequence and mechanistic homology with
Ras proteins.
1 M. Barbacid, Annu. Rev. Biochem. 56, 779 (1987).
~J. L. Bos, Cancer Res. 49, 4682 (1989).
3 L. A. Feig, B. T. Pan, T. M. Roberts, and G. M. Cooper, Proc. Natl. Acad. Sci. USA 83,
4607 (1986).
Copyright ?c( 5991 yb Academic Press. .cnI
SDOHTEM NI .YGOLOMYZNE VOI. 552 All rights of reproduclion ni yna form reserved
4 EXPRESSION, PURIFICATION, AND MODIFICATION 1
Methods
Protein Expression and Cell Growth
The E. coli expression vectors pAT-RasH 4 and pTACC-RasC', 5 encod-
ing the first 166 residues of the human Ras p21 protein Ras p21 (1-166),
have been kindly provided by C. Der and A. Wittinghofer, respectively.
The plasmids are transformed into E. coli strain JM105. Conditions for cell
growth of selectively and uniformly ~SN3C-enriched H-Ras have been
described previously. 76 Ras is expressed by growing bacteria at 33 ° in Luria
broth. At an optical density of -2.3 (600 nm), expression of the protein
is induced by the addition of 1 mM isopropyl-/3-D-thiogalactopyranoside
(IPTG). Samples are collected hourly and the fermentor chilled when the
glucose concentration falls to zero (-4 hr). Cells are harvested by centrifu-
gation at 3300 g, 4 ,° for 30 rain and the cell paste is stored at -80 .° All
subsequent steps are performed at 4 .° The cell paste is resuspended to 0.1
g of cell paste/ml with sonication buffer 20 mM Tris-HC1 (pH 7.2), 100
mM NaC1, 5 mM MgCI2, 1 mM dithiothreitol (DTT), and 1 mM phenyl-
methylsulfonyl fluoride (PMSF) and the cells are washed once by pelleting
at 16,000 g for 10 rain. The cells are resuspended again to 0.1 g of cell
paste/ml with sonication buffer, and then broken by sonication in a 250-
ml Rossett cup (VWR Scientific, Marietta, GA) at maximum output pulsed
50% duty cycle for 45 rain, using a Heat Systems (VWR Scientific, Marietta,
GA) W-375 sonicator equipped with a 0.5-in. button tip. We have also
employed the French press as an alternative method for cell lysis. Soluble
and insoluble fractions are fractionated by centrifugation at 17,000g for 30
rain. If the soluble fraction is not used immediately, ammonium sulfate is
added to 80% saturation, and the resultant mixture is stored at 4 ° . The
insoluble fraction is resuspended to 1.0 vol of sonicated material. All purifi-
cation procedures are performed at 4 ° .
Purification qf Soluble H-Ras Protein
DNA is precipitated from the soluble fraction by the slow addition of
10% polyethyleneimine dissolved in sonication buffer to a final concentra-
tion of 0.03%. It is important that the final concentration of polyethyleneim-
ine does not exceed 0.03%, as Ras protein will start to precipitate at higher
4 C. J. Der. T. Finkel, and G. M. Cooper, (?ell (Cambridge, Mass.) 44, 167 (1986).
J. John, I. Schlichtin, E. Schiltz. P. Rosch. and A. Wininghofer, .J Biol. Chem. 264,
13086 (1989).
'~ P. J. Kraulis, P. J. Domaille, S. L. Campbell-Burk. 3'. Van Aken, and E. Laue, Biochemistry
33, 3515 (1994).
v R. J. DeLoskey, D. E. Van Dyk, T. E. Van Aken. and S. Campbelt-Burk, Arch. Biochem.
Biophys. 311, 72 (1994).
1 REFOLDING AND PURIFICATION OF Ras PROTEINS 5
concentrations. The mixture is then stirred slowly for 02 min and the precipi-
tate pelleted at 27,000 g for 02 min. The resultant supernatant si dialyzed
for 22 hr against 2 × 01 vol of QFF buffer 02 mM Tris-HC1 (pH 0.8 at
4°), 50 mM NaC1, 30/xM GDP, 5 mM ,21CgM %01 glycerol (v/v), and 1 mM
DTT plus 1 mM PMSF. The dialyzed material is then loaded onto a Q-
Sepharose Fast Flow (Pharmacia, Piscataway, N J) anion-exchange column
(4.4 × 14.5 cm) equilibrated with QFF buffer at a flow rate of 4 ml/min.
H-Ras is eluted off the column with a 2-liter gradient of 50-1000 mM NaCI
in QFF buffer. Typically, H-Ras elutes off the column as a broad peak
at 250-450 mM NaC1. The fractions containing H-Ras are pooled and
concentrated to <10 ml using an Amicon (Danvers, MA) stirred cell with
a YM10 membrane.
Gel-filtration chromatography is performed using a Sepharose S-200
high-resolution column (2.5 × 001 cm; Pharmacia) equilibrated with S-200
buffer 02 mM Tris-HCl (pH 8.0, at 4°), 001 mM NaCI, 5 mM ,21CgM 1
mM DTT, %01 (v/v) glycerol, and 30/xM GDP at a flow rate of 2 ml/min.
The fractions containing H-Ras are pooled and concentrated using a YM10
membrane in an Amicon stirred cell and/or a Centricon 01 concentrator
to >20 mg/ml. Western blot analysis and GDP binding are performed on
aliquots from the various purification steps. Concentrated H-Ras protein
is stored at -20 ° after the addition of 6.1 vol of Ras freezing buffer 02
mM Tris-HC1 (pH 8.0), 01 mM NaC1, 5 mM ,21CgM 1 mM DTT, %57
(v/v) glycerol, and 30/xM GDP.
If the soluble fraction is stored as an ammonium sulfate precipitate,
the protein si resuspended with sonication buffer and dialyzed to remove
ammonium sulfate prior to use.
Purification of Guanidine Hydrochloride-Solubilized Ras Protein mo?f.
Inclusion Bodies
The insoluble fraction si resuspended in sonication buffer and pelleted
at 17,000 g, The resultant pellet is resuspended to a protein concentration
of 01 mg/ml with solubilization buffer 5.0 M guanidine hydrochloride, 50
mM Tris-HC1 (pH 8.0), 05 mM NaCI, 5 mM ,21CgM 1 mM EDTA, 5 mM
DTT, 1 mM PMSF, 30/,M GDP, and %5 (v/v) glycerol and stirred for 1
hr. The insoluble material si then pelleted by centrifugation at 17,000 g for
30 min. The supernatant is diluted 100-fold with dilution buffer (same as
solubilization buffer, minus guanidine-HC1 and 1 mM DTT instead of 5
mM DTT) and incubated without stirring for 2 hr. The sample si then
dialyzed against 2 vol of dialysis buffer 02 mM Tris-HCl (pH 8.0), 5 mM
MgCI2, 1 mM DTT, 1 mM PMSF, %5 (v/v) glycerol, and 30/xM GDP for
81 hr. Anion-exchange chromatography is performed using Q-Sepharose
Fast Flow (QFF) resin as described above for the soluble H-Ras protein.
6 EXPRESSION, PURIFICATION, AND MODIFICATION
The QFF fractions are analyzed for GDP-binding activity and by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to deter-
mine which fractions contained H-Ras. The H-Ras fractions are pooled
and concentrated with a YM10 membrane in an Amicon stirred cell to >20
mg/ml and stored at -20 ° after dilution with 2 vol of Ras freezing buffer.
Western blot analysis is performed and GDP-binding activity is measured.
Purification of Urea-Solubilized Ras Protein from Inclusion Bodies
The insoluble fraction resuspended in sonication buffer is pelleted at
17,000 g. The resultant pellet is resuspended to a protein concentration of
01 mg/ml with solubilization buffer 6 M urea, 20 mM Tris-HC1 (pH 8.0),
50 mM NaC1, 5 mM MgC12, 1 mM EDTA, 1 mM 2-mercaptoethanol (2-ME),
1 mM PMSF, 30/xM GDP, and 5% (v/v) glycerol and stirred for 2 hr. The
insoluble material is then pelleted by centrifugation at 17,000 g for 30 min.
The resultant pellet is resuspended to its previous volume with solubilization
buffer and stirred for an additional 2 hr. The insoluble material is then
pelleted by centrifugation at 17,000 g for 30 min. The supernatants from
both spins are combined and diluted 20-fold with dilution buffer 20 mM
Tris (pH 8.0), 50 mM NaC1, 5 mM MgCI2,30/xM GDP, 5% (v/v) glycerol, 1
mM 2-ME and incubated with gentle stirring overnight at 4 .° Alternatively,
solubilized Ras may be dialyzed against the dilution buffer instead of dilut-
ing the sample 20-fold, to remove the urea and allow for refolding. This
alternative procedure reduces the total sample volume for ease of sample
manipulation in subsequent steps. The sample is then spun one more time
to remove insoluble material, and then loaded onto an anion-exchange
chromatography column using QFF resin. The column is washed with one
column volume of QFF buffer 20 mM Tris (pH 8.0), 50 mM NaC1, 5 mM
MgCI2, 30 txM GDP, 10% (v/v) glycerol, 1 mM DTT, then eluted with a
linear salt gradient from 50 to 1000 mM NaC1, over 01 column volumes.
A typical elution profile from the QFF column is shown in Fig. l. The
fractions eluted from the QFF column are analyzed for GDP-binding activ-
ity and by SDS-PAGE to determine which fractions contain H-Ras. The
H-Ras fractions are pooled and concentrated to about 01 ml, using a YM10
membrane in an Amicon stirred cell. The concentrated H-Ras pool is loaded
onto an S-200 gel-filtration column (2.5 × 100 cm) equilibrated with S-200
buffer and eluted at a flow rate of 2.0 ml/min. A representative elution
profile from the S-200 column is shown in Fig. 2. The fractions from the
S-200 column are analyzed by 15% SDS-PAGE gel electrophoresis to
determine where the H-Ras protein has eluted. The fractions containing
H-Ras are pooled and concentrated using a YM10 membrane in an Amicon
stirred cell to >20 mg/ml and stored at -20 ° after dilution with 2 vol of