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 xiii Contributors to Volume 256 Article numbers arc in parentheses following the names of contributors. Affiliations listed are currenl. ARIE ABO (5, 29), Onyx Pharmaceuticals, Richmond, California 94806 PETER ADAMSON (19), Vascular Biology Re- search Centre, Kings College London, Lon- don, W8 7AH, United Kingdom DANIEL E. H. AFAR (15), Department of Mi- crobiology and Molecular Genetics, Univer- sity of California-Los Angeles, Los Angeles, California 90024 SOHAIL AHMED (14), Department of Neuro- chemistry, Institute of Neurology, London WC1N 1PJ, United Kingdom, and Institute of Molecular and Cell Biology, National University of Singapore, Singapore 0511 KLAUS AKTORIES (21), Institute of Pharma- cology and Toxicology, Albert-Ludwigs University, D-79104 Freiburg, Germany PONTUS ASPENSTROM (25), Department of Zoological Cell Biology, Arrhenius Labo- ratories E5, The Wenner-Gren Institute, Stockholm University, S106-91, Sweden DAVID BALTIMORE (17), Massachusetts Insti- tute of Technology, Cambridge, Massachu- setts 02139 PATR1C1A BEROEZ-AULLO (32), Laboratoire de Biologie Mol(culaire et S4quencage, Universit~ Bordeaux II, 33076 Bordeaux, France JACQUES BERTOGLIO (35), INSERM CJF 93- 01, Facultd de Pharmacie-Universit~ Paris- Sud, 92296 Chatenay Malabry Cedex, France GARY M. BOKOCH (4, 28), Departments of Immunology and Cell Biology, The Scripps Research Institute, La Jolla, California 92037 PATR1CE BOQUET (32), Unit~ des Toxines Mi- crobiennes, Institut Pasteur, 75724 Paris, France EDWARD P. BOWMAN (27), Department of. Biochemistry, Emory University Medical School, Atlanta, Georgia 30322 RICHARD A. CERIONE (2, 9, 12), Department of Pharmacology, Cornell University, Ith- aca, New York 14853 PIERA CICCHETTI (17), Institute for Genetics, University of Cologne, Cologne D-50674, Germany DAGMAR DIEKMANN (23), CRC Oncogene and Signal Transduction Group, MRC Lab- oratory for Molecular Cell Biology and Department of Biochemistry, University College London, London WC1E 6BT, United Kingdom SIMON T. DILLON (20), Department of Micro- biology and Molecular Biology, Tufts Uni- versity School qfl Medicine, Boston, Massa- chusetts 02111 OLIVIER DORSEUIL (39), Institut Cochin de G4n4tique Mol&ulaire, 1NSERM UnitO 257, 75014 Paris, France ALESSANDRA EVA (38), Laboratory of Cellu- lar and Molecular Biology, National Cancer Institute, National Institute of Health, Bethesda, Maryland 20892 LARRY a. FEIG (20), Department of Biochem- istry, Tufts University, School of Medicine, Boston, Massachusetts 02111 PHILIPPE FORT (18), Institute of Molecular Ge- netics, University Montipellier, F 340.33 Montpellier, France ROSEMARY FOSTER (13), MGM Cancer Cen- ter and Department of Medicine, Harvard Medical School, Charlestown, Massachu- setts 02129 GERARD GACON (39), Institut Cochin de G~n- (ique Mol~culaire, 1NSERM Unit4 257, 75014 Paris, France MURIELLE GIRY (32), Unit~ des Toxines Mi- crobiennes, Institut Pasteur, 75724 Paris, France IX X CONTRIBUTORS TO VOLUME 256 ALAN HALL (1, 8, 23), MRC Laboratory for Molecular Cell Biology and Department of Biochemistry, University College London, London WC1E 6BT, England CHRISTINE HALL (14), Institute of Neurology, London WC1N 1P J, United Kingdom JOHN F. HANCOCK (10), Onyx Pharmaceuti- cals, Richmond, California 94806 MATTHEW J. HART (9), Department of Phar- macology, Ithaca, New York 14853 DOUGLAS I. JOHNSON (30), Department of Mi- crobiology and Molecular Genetics, Univer- sity of Vermont, Burlington, Vermont05405 INGO JUST (21), Institute of Pharmacology and Toxicology, Albert-Ludwigs University, D-79104 Freiburg, Germany ULLA G. KNAUS (4), Department oflmmunol- DAy, The Scripps Research Institute, La Jolla, California 92037 ROBERT KOZMA (14), Institute of Neurology, London WC1N IPJ, United Kingdom, and Institute of Molecular and Cell Biology, Na- tional University of Singapore, Singapore 0511 J. DAVID LAMBETH (27), Department of BiD- chemistry, Emory University MediCal School, Atlanta, Georgia 30322 PAUL LANG (35), INSERM CJF 93-O1, Facultd de Pharmacie-Universit~ Paris-Sud, 92296 Chatenay Malabry Cedex, France GI~RALD LECA (39), INSERM Unit~131, Association Chlude Bernard, Institute d'Hematologie-HOpital Saint-Louis, Paris, France EMMAUEL LEMICHEZ (32), Unit~ des Toxines Microbiennes, Institut Pasteur, 75724 Paris, France DAVID LEONARD (2,12), Department of Phar- macology, Cornell University, Ithaca, New York 14853 THOMAS LEUNG (16, 24), Institute of Molecu- lar and Cell Biology, National University of Singapore, Singapore 0511 Louis LIM (14, 16, 24), Institute of Neurology, London WC1N 1PJ, United Kindgom, and Institute of Molecular and Cell Biology, Na- tional University of Singapore, Singapore 0511 EDWARD MANSER (16, 24), Institute of Molec- ular and Cell Biology, National University of Singapore, Singapore 0511 JANET MCCULLOUGH (30), Department of Mi- crobiology and Molecular Genetics, Univer- sity of Vermont, Burlington, Vermont 05405 TORU MIKI (11), Laboratory of Cellular and Molecular Biology, National Cancer Insti- tute, National Institutes of Health, Bethesda, Maryland 20892 PETER J. MILLER (30), Department of Micro- biology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405 TAKAKAZU MIZUNO (3), Department of Mo- lecular Biology and Biochemistry, Osaka University Medical School, Suita, Osaka 565, Japan NARITO MORII (22), Department of Pharma- cology, Kyoto University Faculty of Medi- cine, Kyoto 606, Japan HIROYUKI NAKANISHI (3), Department of Mo- lecular Biology and Biochemistry, Osaka University Medical School, Suita, Osaka 565, Japan SHUH NARUMIYA (22, 31), Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto University, Kyoto 606, Japan MICHAEL F. OLSON (25), CRC Oncogene and Signal Transduction Group, MRC Labora- tory for Molecular Cell Biology, University College London, London WCIE 6BT, United Kingdom Huort PATERSON (19), Section of Cell and Molecular Biology, Chester Beatty Labora- tories, Institute of Cancer Research, London SW3 6B J, United Kingdom MARK R. PHILIPS (7), Departments of Medi- cine and Cell Biology, New York University School of Medicine, New York, New York 10016 MICHAEL H. PILLINGER (7), Department of Medicine, New York University School of Medicine, New York, New York 10016 MICHEL R. POPOFF (32), Unit~ des Toxines Microbiennes, Institut Pasteur, 75724 Paris, France CONTRIBUTORS TO VOLUME 256 xi EMILIO PORFIRI (10), Onyx Pharmaceuticals, Richmond, California 94806 JAMES POSADA (30), Department of Microbi- ology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405 MARK T. QUINN (28), Veterinary Molecular Biology, Department of Microbiology, Montana State University, Bozeman, Mon- tana 59717 ANNE J. RIDLEY (33, 3,4), Ludwig Institute for Cancer Research, London WCIP 8BT, United Kingdom SUSAN E. RITTENHOUSE (26), Jefferson Can- cer Institute and Cardeza Foundation for Hematologic Research, Philadelphia, Penn- sylvania 19107 DAVID ROBERTSON (19), Haddow Labora- tories, Institute of Cancer Research, Sutton, Surrey, SM2 5NG, United Kingdom TAKUYA SASAKI (6, 37), Department of Mo- lecular Biology and Biochemistry, Osaka University Medical School, Suita, Osaka 565, Japan ANTHONY W. SEGAL (29), Division of Molec- ular Medicine, University College London, London WCIE 6J J, United Kingdom ANNETTE J. SELF (1, 8), MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, United Kingdom JEFFREY SETTLEMAN (13), MGH Cancer Cen- ter and Department of Medicine, Harvard Medical School, Charlestown, Massachu- setts 02129 MARIE-JOSE STASlA (36), Laboratoire d'En- zymologie, Centre Hospitalier Universitaire de Grenoble, Grenoble, France YOSHIMI TAKAI (3, 6, 37), Department of Mo- lecular Biology and Biochemistry, Osaka University Medical School, Osaka 565, Ja- pan, and Department of Cell Physiology, National Institute for Physiological Sci- ences, Okagaki 444, Japan KENJI TAKAISHI (37), Department of Molecu- lar Biology and Biochemistry, Osaka Uni- versity Medical School Suita 565, Japan KAZUMA TANAKA (6), Department of Molec- ular Biology and Biochemistry, Osaka Uni- versity Medical School Suita, Osaka 565, Japan TOMOKO TOMINAGA (31), Department of Cel- lular and Molecular Physiology, National Institute for Physiological Sciences, Oka- zaki 444, Japan DAVID J. UHLINGER (27), Department of Biochemistry, Emory University Medical School Atlanta, Georgia 30322 A1ME VASQUEZ (39), 1NSERM Unit 131, As- sociation Claude Bernard Research Center, 92140 Clamart, France PIERRE V. VIGNAIS (36), Laboratoire de Bio- chimie, Departement de Biologie Molecu- laire et Structurale, CEA CEN-Grenoble, F-38054 Grenoble, France SYLVIE VINCENT (18), Institute of Molecular Genetics, University Montipellier, F 34033 Montpellier, France OWEN N. WITrE (15), Molecular Biology In- stitute and Howard Hughes Medical Insti- tute, University of California-Los Angeles, Los Angeles, California 90024 DANIELA ZANGR1LLI (38), Laboratory of Cel- lular and Molecular Biology, National Can- cer Institute, National Institutes of Health, Bethesda, Maryland 20892 Y1 ZHENG (2, 9), Department of Pharma- cology, Cornell University, Ithaca, New York 14853 MICHAEL ZIMAN (30), Department of Molecu- lar and Cell Biology, University of Califor- nia, Berkeley, Berkeley, California 94720 [ 1 ] Rho/Rac/G25K FROM 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. 1'2 A wide range of expression levels has been reported for Ras- related proteins in E. coli; in the case of Ras, yields of 7.5 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. 4-9 These proteins are 30% 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 85% 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. 12, 3138 (1992). 10 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 EXPRESSION AND PURIFICATION [ 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 13 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.). 16 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 11M. Katayama, M. Kawata, Y. Yoshida, H. Horiuchi, T. Yamamoto, Y. Matsuura, and Y. Takai, J. Cell. Biol. 266, 12639 (1991). 12 B. T. Kinsella, R. A. Erdman, and W. A. Maltese, J. Biol. Chem. 15, 9786 (1991). 13 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, 286 (1991). a4 p. Adamson, C. J. Marshall, A. Hall, and P. A. Tilbrook, J. Biol. Chem. 267, 20033 (1992). 15H. F. Paterson, A. J. Self, M. D. Garrett, I. Just, K. Aktories, and A. Hall, J. Cell Biol. 111, 1001 (1990). 16 D. B. Smith and K. S. Johnson, Gene 67, 31 (1988). [1] Rho/Rac/G25K FROM E. coli 5 b THROMBIN ILeu Val Pro Arg~Gly serlpro GIy lie His Arg Asp GST CTG G-I-F CCG CGT GGA TCC CCG GGA ATT CAT CGT GAC TGA CTG ACG I I I I BamHl [__] EcoRl Stop codons Smal GST CTG G-I-r CCG CGT GGA TCC CCG GCT rhoA GST CTG GTT CCG CGT GGA TCC CCG CAG., racl GST CTG GTT CCG CGT GGS TCC CCG 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, and 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 37 °, the culture is diluted 1 : 10 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 0.1 mM (0.5 ml of a 0.1 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 10 min at 4 ° and then resuspended (on ice) in 3 ml of cold lysis buffer [50 mM Tris-HC1, pH 7.6, 50 mM NaC1, 5 mM MgC12, 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. 16 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 150 sonicator at an amplitude of 14 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 10 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 [50 mM Tris-HC1, pH 8.0, 150 mM NaC1, 5 mM MgC12, 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 FROM 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 [3H]GTP/[3H]GDP 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 [3H]GTP or [3H]GDP 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 qmlllP 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 = 106 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 [...]... with buffer A, and < /b> resuspended with 10 ml of buffer A Both fractions are stored at -80 ° and < /b> are stable for at least several months CM-Sepharose Column Chromatography One-third of the cytosol fraction (10 ml, 30 mg of protein) is diluted fivefold with buffer B and < /b> applied to a CM-Sepharose column (1.5 x 20 cm) equilibrated with buffer B Elution is performed with 50 ml of buffer B followed by buffer B. .. shown that both forms of Rac are equally effective.21'= The reason for this discrepancy is currently unknown 25A Abo, M R Webb, A Grogan, and < /b> A Segal, Biochem J 298, 585 (1994) 26T Sasaki, M Kato, and < /b> Y Takai, J Biol Chem 268, 23959 (1993) [4] P u r i f i c a t i o n of Rac2 from Human Neutrophils By ULLA G KNAUS and < /b> GARY M BOKOCH The Rac2 protein belongs to the Rho family of GTP-binding proteins and < /b> is... shown that both forms of Rac are equally effective.21'= The reason for this discrepancy is currently unknown 25A Abo, M R Webb, A Grogan, and < /b> A Segal, Biochem J 298, 585 (1994) 26T Sasaki, M Kato, and < /b> Y Takai, J Biol Chem 268, 23959 (1993) [4] P u r i f i c a t i o n of Rac2 from Human Neutrophils By ULLA G KNAUS and < /b> GARY M BOKOCH The Rac2 protein belongs to the Rho family of GTP-binding proteins and < /b> is... hydroxyapatite, and < /b> Mono Q chromatographies 1 Based on immunological cross-reactivity, the bovine brain 22-kDa GTP-binding protein/phosphosubstrate represents a form of the Gp (G25K) protein that was originally identified in human placenta and < /b> platelet plasma membranes 2,3 Two cDNAs encoding this GTP-binding protein have been cloned from human cDNA libraries: one from a human placental library 4 and < /b> the other... protein with a Mr of about 22,000 and < /b> is identified to be Rac2 by the partial amino acid sequences Assay for Cell-Free NADPH Oxidase Activity The cell-free NADPH oxidase activity is assayed by measuring the arachidonic acid-elicited superoxide generation, which is determined by the SOD-inhibitable ferricytochrome c reduction by use of Rac2, p47-phox, p67-phox, GTPyS, and < /b> the solubilized membrane components... phenyl-Superose H R 5/5 F P L C column (Pharmacia LKB Biotechnology Inc.) equilibrated with the same 13s Shaltiel, in "Methods in Enzymology" (W B Jakoby and < /b> M Wilchek, eds.), Vol 34, p 126 Academic Press, New York, 1974 [41 PURIFICATION OF R a c 2 FROM NEUTROPHILS 31 buffer, the column is washed with 8 ml of equilibration buffer until the absorbance baseline is obtained The elution is performed with a simultaneous... filter binding assay using [3H]GDP or [3H]GTP b Coomassie blue staining of electrophoresed proteins using B S A as standard c Binding assays with N 1 7 R a c l and < /b> N17G25K carried out using [3H]GDP only 10 EXPRESSIONAND PURIFICATION [ 1] M u t a n t Rho, Rac, and < /b> G25K Proteins We have purified a variety of Rho, Rac, and < /b> G25K proteins containing amino acid substitutions using the pGEX-2T vector and < /b> the... to asparagine substitution at codon 17 in Rac (N17Rac) and < /b> G25K (N17G25K), equivalent to the dominant negative N17 mutation in Ras The yields of these mutant proteins as determined by nucleotide binding and < /b> Coomassie staining of acrylamide gels are shown in Table I Table I shows that the yields, as judged by nucleotide binding of N17Racl, V12G25K, and < /b> particularly N17G25K, are very low but that the actual... described earlier As can be seen from Fig 2, N25Rho migrates slightly slower than wild-type RhoA and < /b> produces a much sharper band We have found that all Ras and < /b> Rho-related GTPases < /b> are prone to smearing after electrophoresis, particularly if freshly prepared sample loading buffer is not used, and < /b> it is likely that the proteins are sensitive to oxidation Even with fresh buffer, however, the smearing observed... source for the purification of biologically active Rac2, if recombinant sources that allow post-translational processing of GTP-binding pro1J Didsbury, R F Weber, G.M Bokoch, T Evans, and < /b> R Snyderman,J BioL Chem 264, 16378 (1989) 2M T Quinn, T Evans, L R Loetterle, A.J Jesaitis, and < /b> G M Bokoch, Z BioL Chem 268, 20983 (1993) METHODS IN ENZYMOLOGY, VOL 256 Copyright © 1995 by Academic Press, Inc All rights . 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. Cdc42Hs fractions can be identified by Western blot analysis using the anti-Cdc42Hs antibody or by Coomassie blue staining (i.e., as indicated by the presence of a single protein band at 22 kDa) Laboratory of Cellular and Molecular Biology, National Cancer Insti- tute, National Institutes of Health, Bethesda, Maryland 20892 PETER J. MILLER (30), Department of Micro- biology and