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. CHANNING J. DER W.E. BALCH ALANHALL Contributors to Volume 255 Article numbers are. in parentheses following the names Affiliations listed are current. of contributors. 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- DAFNA BAR-SAGI (13,43), Cold Spring Har- ogy and Anatomy, University of Virginia bor Laboratory, Cold Spring Harbor, New Health Sciences Center, Charlottesville, Vir- York II 724 ginia 22908 RHONDA L. BOCK (38) Department of Cancer ANDREW D. CATLING (25), Department of Mi- Research, Merck Research Laboratories, crobiology and Cancer Center, School of West Point, Pennsylvania 19486 Medicine, University of Virginia, Char- GIDEON E. BOLLAG (2,3,18), Onyx Pharma- lottesville, Virginia 22908 ceuticals, Richmond, California 94806 RITA S. CHA (44), Center for Environmental JOHANNES L. Bos (17, 22) Laboratory for Health Sciences, Massachusetts Institute of Physiological Chemistry, University of Technology, Cambridge, Massachusetts 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, Chapel Hill, North Carolina 27599 LI CHEN (46), Onyx Pharmaceuticals, Rich- 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 Southern California School of Medicine, GEOFFREY J. CLARK (40), Department of Los Angeles, California 90033 Pharmacology, School of Medicine, Univer- 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- western Medical Center, Dallas, Texas PHILIP COHEN (29) MRC Protein Phosphory- 75235 lation Unit, Department of Biochemistry, University of Dundee, Dundee DDI 4HN BOUDEWUN M. T. BURGERING (22) Labora- Scotland ix X CONTRIBUTORS ROBBERT H. COOL (lo), Max-Planck-Institut fiir Molekulare Physiologie, 44139 Dort- mund, Germany GEOFFREY M. COOPER (23), Dana-Farber Cancer Institute and Department of Pathol- ogy, Harvard Medical School, Boston, Mas- sachusetts 02115 SALLY COWLEY (29), Chester Beatty Labora- tories, Institute of Cancer Research, London SW3 6JB, United Kingdom ADRIENNE D. Cox (21, 40), Departments of Radiation Oncology and Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 DIDIER CUSSAC (13), Institutede Pharmacolo- gie Moleculaire et Cellulaire, 06560 Val- bonne, France ALIDA M. M. DE VRIES-SMITS (17,22), Labo- ratory for Physiological Chemistry, Univer- sity of Utrecht, Utrecht, The Netherlands PAUL DENT (27) Howard Hughes Medical Institute, and Markey Center for Signal Transduction, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908 CHANNING J. DER (6,21,40), Department of Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 JULIAN DOWNWARD (11,17), Imperial Cancer Research Fund, London, WC2A 3PX, United Kingdom CHRISTINE ELLIS (20), Institute of Cancer Re- search, Chester Beatty Laboratories, Lon- don SW3 6JB, United Kingdom TONY EVANS (2) Onyx Pharmaceuticals, Richmond, California 94806 STEPHAN M. FELLER (37), Laboratory of Mo- lecular Oncology, Rockefeller University, New York, New York 10021 JEFFREY FIELD (47) Department of Pharma- cology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania I9104 CATHY FINLAY (39), Department of Cell Biol- ogy, Glaxo Inc., Research Triangle Park, North Carolina 27709 TO VOLUME 255 ROBERT FINNEY (32), Molecular Cancer Biol- ogy, Cell Therapeutics, Seattle, Washing- ton 98119 MA~HIAS FRECH (13) Institute de Pharma- cologie Moleculaire et Cellulaire, 06560 Val- bonne, France JACKSON B. GIBBS (12, 19, 38), Department of Cancer Research, Merck Research Labo- ratories, West Point, Pennsylvania 19486 JOSEPH L. GOLDSTEIN (5) Department of Mo- lecular Genetics, University of Texas South- western Medical Center, Dallas, Texas 75235 SUZANNE M. GRAHAM (40), Department of Pharmacology, School of Medicine, Univer- sity of North Carolina at Chapel Hil, Chapel Hill, North Carolina 27599 HIDESABURO HANAFUSA (37) Laboratory of Molecular Oncology, Rockefeller Univer- sity, New York, New York 10021 JOHN F. HANCOCK (2,7,24), Onyx Pharma- ceuticals, Richmond, California 94806 MATT J. HART (14), Onyx Pharmaceuticals, Richmond, California 94806 CRAIG A. HAUSER (41), Cancer Research Center, La Jolla Cancer Research Founda- tion, La Jolla, California 92037 DESIREE HERRERA (32), Molecular Cancer Biology, Cell Therapeutics, Seattle, Wash- ington 98119 STANLEY M. HOLLENBERG (34) Vellum Insti- tute, Portland, Oregon 97201 GUY L. JAMES (5) Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235 MICHEL JANICOT (42), Rhone-Poulenc Rorer, Centre de Recherche de Vitry/Alfortville, 94403 Vitry sur Seine, France ALGIRDAS J. JESAITIS (48) Department of Mi- crobiology, Montana State University, Bozeman, Montana 59717 WEI JIANG (45) Molecular Biology and Virol- ogy Laboratory, The Salk Institute, La Jolla, California 92037 GARY L. JOHNSON (30) Division of Basic Sci- ences, National Jewish Center for Immunol- ogy and Respiratory Medicine, Denver, Colorado 80206, and Department of Phar- CONTRIBUTORS TO VOLUME 255 xi macology, University of Colorado Medical School, Denver, Colorado 80262 J. DEDRICK JORDAN (21), Department of Chemistry, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Scold M. KAHN (45), Center for Radiological Research, Columbia University, New York, New York 10032 BRIAN K. KAY (50) Curriculum in Genetics and Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 YOSHITO KAZIRO (16) Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226, Japan MIREI~LE KENIGSBERG (42), Rhone-Poulenc Rorer, Centre de Recherche de Vitry/Alfort- ville, 94403 Vitry sur Seine, France ROYA KHOSRAVI-FAR (6) Department of Pharmacology, School of Medicine, Univer- sity of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 BEATRICE KNUDSEN (37), Laboratory of Mo- lecular Oncology, Rockefeller University, New York, New York 10021 NANCY E. KOHL (38) Department of Cancer Research, Merck Research Laboratories, West Point, Pennsylvania 19486 SHINYA KURODA (26) Department of Molec- ular Biology and Biochemistry, Osaka Uni- versity Medical School, Okazaki 444, Ja- pan, and Department of Cell Physiology, National Institute for Physiological Sci- ences, Okazaki 444, Japan CAROL A. LANGE-CARTER (30) Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206, and Department of Pharmacology, University of Colorado Medical School, Denver, Colorado 80262 SALLY J. LEEVERS (28, 29), Chester Beatty Laboratories, Institute of Cancer Research, London SW3 6JB, United Kingdom CHRISTIAN LENZEN (lo), Max-Planck-Insti- tute fur Molekulare Physiologie, 44139 Dortmund, Germany BEN MARGOLIS (36), Department of Pharma- cology, and Kaplan Cancer Center, New York University Medical Center, New York, New York 10016 CHRISTOPHER J. MARSHALL (28, 29), Chester Beatty Laboratories, Institute of Cancer Re- search, London SW3 6JB, United Kingdom MARK S. MARSHALL (33) Department of Medicine, Division of Hematology and On- cology, and Walther Oncology Center, Indi- ana University, Indianapolis, Indiana 46202 FRANK MCCORMICK (3, 18), Onyx Pharma- ceuticals, Richmond, California 94806 VIVIEN MEASDAY (20), Banting and Best De- partment of Medical Research, University of Toronto, Toronto, Canada M5G IL6 ANDREI MIKHEEV (44), Center for Environ- mental Health Sciences, Massachusetts Insti- tute of Technology, Cambridge, Massachu- setts 02139 KEITH A. MINTZER (47), Department of Phar- macology, University of Pennsylvania School of Medicine, Philadelphia, Pennsyl- vania 19104 HIROSHI MITSUZAWA (9), Department of Mi- crobiology and Molecular Genetics, Univer- sity of California at Los Angeles, Los Angeles, California 90024 MICHAEL F. MORAN (20), Banting and Best Department of Medical Research, Univer- sity of Toronto, Toronto, Canada MSG I L6 DEBORAH K. MORRISON (31), Cellular Growth Mechanisms Group, ABL-Basic Research Program, NCI-FCRDC, Freder- ick, Maryland 21702 SCOTT D. MOSSER (38) Department of Cancer Research, Merck Research Laboratories, West Point, Pennsylvania 19486 RAYMOND D. MOSTELLER (15), Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, Uni- versity of Southern California School of Medicine, Los Angeles, California 90033 ALLEN OLIFF (38) Department of Cancer Re- search, Merck Research Laboratories, West Point, Pennsylvania, 19486 WEONMEE PARK (15), Department of Biologi- cal Sciences, Molecular Biology Program, xii CONTRIBUTORS TO VOLUME 255 University of Southern California, Los Angeles, California 90089 CHARLES A. PARKOS (48), Department of Pa- thology, Brigham and Women’s Hospital, Boston, Massachusetts 02115 MANUEL PEIwCHO (45), California Institute of Biological Research, La Jolla, Califor- nia 92037 PAUL POLAKIS (A), GnyX Pharmaceuticals, Richmond, California 94806 EMILIO PORFIRI (2), Onyx Pharmaceuticals, Richmond, California 94806 PATRICK POULLET (49), Department of Micro- biology and Molecular Genetics, University of California at Los Angeles, Los Angeles, California 90024 Scan POWERS (14, 46) Onyx Pharmaceuti- cals, Richmond, California 94806 LAWRENCE A. QUILLIAM (41,50), Department of Pharmacology, University of North Car- olina at Chapel Hill, Chapel Hill, North Carolina 27599 MARK T. QUINN (48) Veterinary Molecular Biology, Montana State University, Boze- man, Montana 59717 CHRISTOPH W. M. REUTER (25), Department of Microbiology and Cancer Center, School of Medicine, University of Virginia, Char- lottesville, Virginia 22908 GUILLERMO ROMERO (27) Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 BONNEE RUBINFELD (4), Onyx Pharmaceuti- cals, Richmond, California 94806 TAKAYA SATOH (16), Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226, Japan MICHAEL D. SCHABER (19) Department of Cancer Research, Merck Research Labora- tories, West Point, Pennsylvania 19486 JOSEPH SCHLESSINGER (36) Department of Pharmacology, New York University, Med- ical Center, New York, New York 10016 KAZUVA SHIMIZU (26) Department of Molec- ular Biology and Biochemistry, Osaka Uni- versity Medical School, Okazaki 444, Ja- pan, and Department of Cell Physiology, National Institute for Physiological Sci- ences, Okazaki 444, Japan EDWARD Y. SKOLNIK (36) Departments of Pharmacology and Internal Medicine, Skir- ball Institute for Biomolecular Medicine, New York University Medical Center, New York, New York 10016 PATRICIA A. SOLSKI (21), Department of Pharmacology, School of Medicine, Univer- sity of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 ANDREW B. SPARKS (50) Curriculum in Ge- netics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 JEFFRY B. STOCK (8), Departments of Molecu- lar Biology and Chemistry, Lewis Thomas Laboratory, Princeton University, Princeton, New Jersey 08544 THOMAS W. STIJRGILL (27) Howard Hughes Medical Institute, and Markey Center for Signal Transduction, University of Virginia Health Sciences Center, Charlottesville, Vir- ginia 22908 YOSHIMI TAKAI (26) Department of Molecu- lar Biology and Biochemistry, Medical School, Osaka University, Osaka 565, Japan FUYUHIKO TAMANOI (9, 49) Department of Microbiology and Molecular Genetics, Uni- versity of California at Los Angeles, Los Angeles, California 90024 TRAC( J. THOMAS (38) Department of Cancer Research, Merck Research Laboratories, West Point, Pennsylvania 19486 JUDITH M. THORN (50) Department of Biol- ogy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 BRUNO TOCQUE (42), Rhone-Poulenc Rorer, Centre de Recherche de Vitry/Alfortville, 94403 Vitry sur Seine, France LOESJE VANDERVOORN (17),Laboratory for Physiological Chemistry, University of Utrecht, Utrecht, The Netherlands ANNE B. VOITEK (34) Fred Hutchinson Can- cer Research Center, Seattle, Washington 98104 CRAIG VOLKER (8), Departments of Molecu- 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 [i] REFOLDING AND PURIFICATION OF Ras 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 is produced using the expression system of Feig et al., 3 95-99% is localized in the inclusion bodies as insoluble protein, whereas 1-5% is 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 is 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? 1995 by Academic Press. Inc. METHODS IN ENZYMOLOGY. VOI. 255 All rights of reproduclion in any 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 ~SN]3C-enriched H-Ras have been described previously. 67 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 0.1 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 20 min and the precipi- tate pelleted at 27,000 g for 20 min. The resultant supernatant is dialyzed for 22 hr against 2 × 10 vol of QFF buffer [20 mM Tris-HC1 (pH 8.0 at 4°), 50 mM NaC1, 30/xM GDP, 5 mM MgC12, 10% 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 × 100 cm; Pharmacia) equilibrated with S-200 buffer [20 mM Tris-HCl (pH 8.0, at 4°), 100 mM NaCI, 5 mM MgC12, 1 mM DTT, 10% (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 10 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 1.6 vol of Ras freezing buffer [20 mM Tris-HC1 (pH 8.0), 10 mM NaC1, 5 mM MgC12, 1 mM DTT, 75% (v/v) glycerol, and 30/xM GDP]. If the soluble fraction is stored as an ammonium sulfate precipitate, the protein is resuspended with sonication buffer and dialyzed to remove ammonium sulfate prior to use. Purification of Guanidine Hydrochloride-Solubilized Ras Protein .f?om Inclusion Bodies The insoluble fraction is resuspended in sonication buffer and pelleted at 17,000 g, The resultant pellet is resuspended to a protein concentration of 10 mg/ml with solubilization buffer [5.0 M guanidine hydrochloride, 50 mM Tris-HC1 (pH 8.0), 50 mM NaCI, 5 mM MgC12, 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 is 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 is then dialyzed against 2 vol of dialysis buffer [20 mM Tris-HCl (pH 8.0), 5 mM MgCI2, 1 mM DTT, 1 mM PMSF, 5% (v/v) glycerol, and 30/xM GDP] for 18 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 10 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 10 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 10 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 [...]... differentiation 1 Mutation of the ras genes, resulting in amino acid changes at positions 12, 13, or 61, can trigger neoplastic transformation and has been detected in about 20% of all human tumors Rap proteins (Rap 1A, Rap l B, and Rap2) are Ras-related GTPases that share 53% amino acid homology with Ras and are able to antagonize the effects of oncogenic Ras in v i v o 2 1 H R Bourne D A Sanders, and. .. posttranslational modifications occurring at the carboxy-terminal C A A X motif (C, cysteine; A, aliphatic; X, any amino acid)) These modifications comprise farnesylation (Ras) or geranylgeranylation (Rap) of the cysteine residue, removal of the A A X amino acids, and c a r b o x y m e t h y l a t i o n Y In addition H-Ras, N-Ras, and K-Ras (A) require palmitoylation, whereas K-Ras(B), which is not palmitoylated,... protein values for full-length Ras calculated from the Bio-Rad assay and from amino acid analysis should be the same However, the protein value for truncated Ras calculated from the Bio-Rad assay is 1.15-fold higher than the value obtained by amino acid analysis S D S - P A GE and Gel Scanning S D S - P A G E is performed using precast Daiichi 10-20% polyacrylamide gels purchased from Integrated Separation... using standard 15% polyacrylamide gels and buffers reported by Laemmli ') Bio-Rad low-range molecular weight standards are used as molecular weight markers Gels are scanned using an LKB (Bromma, Sweden) Ultroscan XL laser densitometer or a Molecular Dynamics (Sunnyvale, CA) computing densitometer and the data are processed using GelScan XL version 1.2 software or ImageQuant version 3.15 software Guanine... K Kaibuchi, T Yamamoto, M Kawamura, T Sakoda, H Fujioka, Y Matsuura, and Y Takai Proc Natl Acad Sci U.S .A 88, 6442 (1991) [2] PURIFICATION OF BACULOVIRUS-EXPRESSED Ras AND Rap 15 and cysteine methylation 11 It has been estimated that processed Ras can constitute up to 20% of the total Ras protein expressed in insect cells, s Ion-exchange chromatography on Mono Q, followed by gel-filtration chromatography... interest and a suitable assay for monitoring relatively weak increases in GTPase activity on addition of a dilute sample of unpurifled GAP A G A P specific for the Ras relative p21 RaN was purified from a variety of mammalian sources I 3 The purified Rapl GAP, which ultimately led to the cloning of the c D N A , was extracted from naembrane fractions prepared from bovine brain tissue Rap1 G A P prepared... _~2M Spaargaren G A Martin, F McCormick, M J Fernandez-Sarabia and J R Bischoff 13iochern l 300, 303 (1994) [3] P u r i f i c a t i o n o f R e c o m b i n a n t R a s GTPase-Activating Proteins By GIDEON BOLLAG and FRANKMcCORMICK Introduction Deactivation of Ras GTP is achieved by catalyzed GTP hydrolysis LIn human tissues, at least two proteins are capable of catalyzing this hydrolysis on Ras, and they... H-Ras is farnesylated and palmitoylated, s K-Ras is farnesylated, ~ )and Rap is geranylgeranylated, u~The series of posttranslational modifications is completed by proteolysis of the A A X amino acids 3 B M Willumsen K Norris, A G Papageorge, N L Hubbert, and D R Lowy EMBO J 3, 2581 (1984) 4j F Hancock, A 1 Magee, J E Childs, and C J Marshall, Cell (Cambridge, Mass.) 57, 1167 (1989) 5 S Clarke, Annu Rev... R a p l G A P originally purified from particulate fractions Moreover, a G A P that stimulated the GTPase activity of the Rap2 protein was also purified, but again was determined to be a 55-kDa degradation product of the 85- to 95-kDa membrane Rapl GAP 6 This 55-kDa form retains activity approximately equivalent to that of the full-length 85to 95-kDa form of Rapl GAP The molecular cloning of the R a. .. the plasmids that express full-length and truncated H-Ras, respectively [2] P u r i f i c a t i o n of Baculovirus-Expressed Ras and Rap Proteins Recombinant B y EMILIO PORF1R1, TONY EVANS, GIDEON BOLLAG, ROBIN CLARK, and JOHN F HANCOCK Introduction H-Ras, N-Ras, K-Ras (A) , and K-Ras(B) are membrane-bound guanine nucleotide-binding proteins that participate in the regulation of cell proliferation and . all human tumors. Rap proteins (Rap 1 A, Rap l B, and Rap2) are Ras-related GTPases that share 53% amino acid homology with Ras and are able to antagonize the effects of oncogenic Ras in. modifications comprise farnesylation (Ras) or geranyl- geranylation (Rap) of the cysteine residue, removal of the AAX amino acids, and carboxymethylationY In addition H-Ras, N-Ras, and K-Ras (A) . in the baculovirus-insect cell system are processed in the same way as in mammalian cells. H-Ras is farnesylated and palmitoylated, s K-Ras is farnesylated, ~) and Rap is geranylgeranylated,