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Purification of Heterotrimeric G Protein   Subunits by GST-Ric-8 Association: PRIMARY CHARACTERIZATION OF PURIFIED G olf

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Purification of Heterotrimeric G Protein   Subunits by GST-Ric-8 Association: PRIMARY CHARACTERIZATION OF PURIFIED G olf
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   1 Purification of Heterotrimeric G protein Subunits by GST-Ric-8 Association: Primary Characterization of Purified G   olf*. PuiYee Chan 2 , Meital Gabay 2 , Forrest A. Wright 2 , Wei Kan  , Sukru S. Oner ¤ , Stephen M. Lanier ¤ , Alan V. Smrcka  , Joe B. Blumer ¤ , and Gregory G. Tall 1    From Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester,  NY 14642 and   ¤ Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425   Running title: G protein !  subunit purification. 1 Address Correspondence to: Gregory G. Tall, Ph.D., 601 Elmwood Ave., Box 711, Rochester, NY 14642. Fax: 585-275-2652; E-mail: gregory_tall@urmc.rochester.edu  2 These authors contributed equally to this work. Ric-8A and Ric-8B are non-receptor G protein guanine nucleotide exchange factors that collectively bind the four subfamilies of G protein subunits. Co-expression of G   subunits with Ric-8A or Ric-8B in HEK293 cells or insect cells greatly promoted G   protein expression. We exploited these characteristics of Ric-8 proteins to develop a simplified method for recombinant G protein subunit purification that was applicable to all G   subunit classes. The method allowed production of the olfactory adenylyl cyclase stimulatory protein, G   olf for the first time, and unprecedented yield of G   q and G   13. G   subunits were co-expressed with GST-tagged-Ric-8A or -Ric-8B in insect cells. GST-Ric-8:G   complexes were isolated from whole cell detergent lysates with glutathione Sepharose. G   subunits were dissociated from GST-Ric-8 with GDP-AlF 4-  (GTP mimicry) and found to be >80% pure, bind GTP   S, and stimulate appropriate G protein effector enzymes. A primary characterization of G   olf showed that it binds GTP   S at a rate marginally slower than G   s s  and directly activates adenylyl cyclase isoforms 3, 5, and 6 with less efficacy than G   s s . Heterotrimeric guanine nucleotide binding  proteins (G proteins) are the foremost signal-transducing molecules used by G-protein-coupled-receptors (GPCRs) to regulate sensation and cellular physiology. Agonist-stimulated GPCRs are guanine nucleotide exchange factors (GEFs) that stimulate G protein !  subunit (G ! ) GDP release. Subsequent GTP binding to G !  causes heterotrimer dissociation or re-arrangement so that G ! -GTP and G #"  adopt states for efficient activation of downstream effector enzymes. Purified G protein subunits have been essential reagents used to develop the current understanding of G protein function, structure, and signaling  pathways (1,2). Current knowledge of traditional G protein signaling network complexity is expanding, and G proteins have been assigned new non-traditional signaling roles including regulation of cell division through unique classes of effector and modulatory enzymes (3-5). As cross-disciplinary G protein research proliferates, the need for purified components to elucidate G  protein functionality is significant. G protein heterotrimers are classified by the identity of the guanine nucleotide-binding subunit: G ! . There are four classes of G !   subunits, G ! s, G ! i, G ! q, and G ! 12/13. Efficient procedures are in place to produce most G ! i-class subunits and G ! s from  E. coli (6,7). Members of the G ! q and G ! 12/13 classes can be prepared from an insect cell expression system using a G #"  co-purification  procedure. This method involves tagging the G "  subunit with a His 6 -tag, isolating the trimeric G  protein by metal chelate chromatography, and elution of the G !  with high specificity using GTP mimicry. This method is tried and true, but rather laborious and involves extensive steps of cell membrane preparation, washing, and detergent extraction. The procedure also results in low G !  yields ( !  50-200 " g protein / L of cell http://www.jbc.org/cgi/doi/10.1074/jbc.M110.178897The latest version is at JBC Papers in Press. Published on November 29, 2010 as Manuscript M110.178897   Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.   b  y g u e  s  t   on J   a n u a r  y 9  ,2  0 1  7 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  b  y g u e  s  t   on J   a n u a r  y 9  ,2  0 1  7 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  b  y g u e  s  t   on J   a n u a r  y 9  ,2  0 1  7 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  b  y g u e  s  t   on J   a n u a r  y 9  ,2  0 1  7 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  b  y g u e  s  t   on J   a n u a r  y 9  ,2  0 1  7 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  b  y g u e  s  t   on J   a n u a r  y 9  ,2  0 1  7 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  b  y g u e  s  t   on J   a n u a r  y 9  ,2  0 1  7 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  b  y g u e  s  t   on J   a n u a r  y 9  ,2  0 1  7 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  b  y g u e  s  t   on J   a n u a r  y 9  ,2  0 1  7 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  b  y g u e  s  t   on J   a n u a r  y 9  ,2  0 1  7 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om    2 culture) (8-11). To our knowledge, the prime target of the largest class of GPCRs, olfactory-specific G ! olf (a G ! s-family member) has not  been purified in sufficient, active quantity to  permit its characterization. While characterizing the G protein GEF activity of Resistance to Inhibitors of Cholinesterase 8 (Ric-8), a series of observations were made that led us to hypothesize that Ric-8  proteins could be used as molecular tools to  prepare recombinant G !  subunits; 1. Ric-8A and Ric-8B collectively bound all four G !  subunit classes (12,13), 2. The Ric-8A:G !  interaction could be manipulated with guanine nucleotides; G ! i 1 formed a stable complex with GST-Ric-8A in the presence of GDP, but was dissociated by GTP( " S) (13,14), 3. Reduction of Ric-8 expression through genetic interventions reduced  plasma-membrane localization of different G !  subunits (15-19), implying that Ric-8 proteins may  positively affect G protein expression. 4. Ric-8B transfection in mammalian cells promoted G ! s/G ! olf expression (18,20). Here we introduce a method for G !  subunit  purification that substantially improves upon established methods in its simplicity, uniformity of application towards all G !  subunit classes, and yield and purity of G protein obtained. Co-expression of GST-tagged Ric-8A or B and G !  subunits in insect cells permitted the isolation of GST-Ric-8:G !  complexes from whole cell detergent lysates with glutathione Sepharose. G !  subunits were recovered specifically from this matrix by elution with AlF 4-  and desalted. This  procedure allowed the first production of active G ! olf, an olfactory/brain-specific stimulator of adenylyl cyclase. Using in vitro effector enzyme reconstitution assays we show that the G !   subunits produced by these means are functional  proteins and demonstrate that G ! olf is a less  potent and efficacious activator of adenylyl cyclase isoforms than equivalently produced G ! s s . EXPERIMENTAL PROCEDURES Quantitative   G !  -YFP expression assaysÐР HEK293 cells were co-transfected as described (21) with pcDNA3.1-G ! i 1 -YFP (22), or  pcDNAI/Amp-G ! s-YFP (a gift from Dr. Catherine H. Berlot, Geisinger Health System, Danville, PA) (23) and pcDNA3.1 constructs that expressed Ric-8A (13), or Ric-8BFL, or Ric-8B $ 9. Fluorescence measurements were  performed as described previously (21). Forty-eight hours after transfection, the cells were harvested with TyrodeÕs solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 0.37 mM  NaH 2 PO 4 , 24 mM NaHCO 3 , 10 mM HEPES-KOH  pH 7.4, and 0.1% glucose (m/v)) and distributed in triplicate at 1 x 10 5  cells/well into gray 96-well  plates. Total fluorescence (excitation Ð 485 nm, emission - 535 nm) was measured to quantify G ! i 1 -YFP or G ! s-YFP expression using a TriStar LB 941 plate reader (Berthold Technologies, Oak Ridge, TN). Data are plotted in relative fluorescent units and are the average of 3 independent transfection experiments.  Insect cell culture and protein expressionÐР GST-tagged Ric-8A and untagged G protein !  subunit baculoviruses were described previously (8-11,13,24). A GST-Ric-8B baculovirus-targeting construct was created using linker-based PCR to amplify full-length mouse Ric-8B from a  purchased clone (Invitrogen, Inc. LLAM collection clone # 6490136 in pCMV-Sport6). The amplified product was digested and ligated into the  Eco RI and Sal I restriction sites of  pFASTBac GST-TEV(13). The resultant amino acid sequences of the tagged Ric-8 proteins were  NÕ-GST- TEV site -Glu(E)-Phe(F)-Ric-8-CÕ. If cleaved by TEV protease digestion, the sequences  become NÕ-Gly(G)-Glu(E)-Phe(F)-Ric-8-CÕ. Recombinant baculoviruses were produced after transfection of adherent Sf  9 cells per the manufacturerÕs instructions (Bac-to-Bac system, Invitrogen). The transfection viral medium supernatants were harvested after nine days and 1/100th culture volumes were amplified twice for five days in log phase Sf  9 suspension cells grown in shake flasks at 2.0 x 10 6  cells/ml. Suspension Sf  9 cells were grown in IPL41 medium containing 10% v/v heat inactivated FBS. Secondarily amplified viruses (5-10 ml of GST-Ric-8 and 5-15 ml of G ! ) were used to co-infect 1L High-five insect cell cultures growing at 2.0 x 10 6  cells/ml in Sf900II medium (Invitrogen). After 48 h expression, cells were harvested by centrifugation at 2000 x g and stored as a cell paste at -80 ¼C until use. The optimal amounts and ratios of secondary amplified viruses used were determined  b  y g u e  s  t   on J   a n u a r  y 9  ,2  0 1  7 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om    3 empirically in smaller-sized culture (50-200 ml)  prior to conducting large-scale (1L) preparations (Figure 1S).  Hi-trap Q anion exchange ChromatographyÐР High-five insect cells (200 ml) grown in suspension to a density of 2.0 x 10 6  cells/ml were infected with 1/100 th  volumes of twice amplified GST-Ric-8A and/or G ! q baculovirus stocks for 48 h. The cell pellets were collected by centrifugation at 1500 x g and lysed in 200 ml of Buffer N (20 mM HEPES-KOH, pH 8.0, 2 mM MgCl 2 , 1 mM EDTA, 1 mM DTT, 11 mM CHAPS, and protease inhibitor mixture) by Parr  bomb nitrogen cavitation. The detergent whole cell lysates were clarified by centrifugation at 100,000 x g for 45 min, passaged through a 0.22 " m filter and loaded onto a 5 ml Hi trap Q column at 1 ml/min using a BioRad Duoflow system. The column was washed with Buffer N and eluted with a linear gradient to 500 mM NaCl in Buffer N. Fractions of the eluate were collected as the gradient developed. Fractions containing G ! q were analyzed by Western blot with anti-G ! q/11 antibody, C-19 (Santa Cruz, Inc. SC-392), Coomassie-stained SDS polyacrylamide gel analysis, and by the GTP " S nitrocellulose filter  binding assay. Glutathione Sepharose ChromatographyÐР Cell  pastes were suspended in 300 ml of detergent lysis  buffer [20 mM HEPES-KOH pH 8.0, 150 mM  NaCl, 1 mM DTT, 1 mM EDTA, 0.05% m/v Genapol C100 detergent (Calbiochem), containing  protease inhibitor mixture; (23 " g/ml  phenylmethylsulfonyl fluoride, 21 " g/ml Na-  p -tosyl-L-lysine-chloromethyl ketone (TLCK), 21 " g/ml L-1-  p -tosylamino-2-phenylethyl-chloromethyl ketone (TPCK), 3.3 " g/ml leupeptin, and 3.3 " g/ml lima bean trypsin inhibitor)] and stirred at 4 ¼C for 30 min. The detergent lysates were homogenized/disrupted by nitrogen cavitation using a Parr Bomb (Parr Instrument, Moline, IL), or by tight pestle dounce homogenization (Kontes, Vineland, NJ). The lysates were centrifuged sequentially at 3000 x g for 10 min and 100,000 x g for 45 min. The clarified detergent-supernatants were loaded onto  packed 5 mL bed volume glutathione Sepharose 4B columns driven by gravity. The column flow through was reapplied to this matrix one time. The columns were washed with 20 column volumes of CHAPS buffer (20 mM HEPES-KOH  pH 8.0, 100 mM NaCl, 1 mM DTT, 11 mM CHAPS, and protease inhibitor mixture) and then warmed to 22 ¼C. To elute G !  subunits, 50 ml of 30 ¼C AMF buffer (20 mM HEPES-KOH pH 8.0, 100 mM NaCl, 50 mM MgCl 2 , 1 mM DTT, 10 mM NaF, 30 " M AlCl 3 , 11 mM CHAPS, and 100 " M GTP) was applied to the columns and allowed to flow through slowly. G !  subunits were typically eluted in the first 10-15 ml with this elution buffer. Ric-8 proteins were then eluted with CHAPS buffer containing 20 mM reduced glutathione. G !  yield was measured by Bradford assay and purity estimated by IMAGE J (v.10.2) analysis of full Coomassie-stained SDS- polyacrylamide gel lanes.  PD-10-desalting gel filtrationÐР AlF 4-  and excess MgCl 2  removal could be accomplished by  passaging G !  subunits through PD-10 desalting columns (GE Healthcare). G !  subunits in AMF  buffer were concentrated in Vivaspin-20 30,000 MWCO ultrafiltration centrifugal concentrators (Sartorius Stedim Biotech, Goettingen, Germany) to a final volume of 2.5 ml (no more than 5 mg/ml  protein) and passaged onto a PD-10 column pre-equilibrated with CHAPS storage buffer (20 mM HEPES-KOH, pH 8.0, 1 mM DTT, 0.5 mM EDTA, 1 " M GDP, and 11 mM CHAPS). G !  subunits were eluted by gravity in 3.5 ml of storage buffer and concentrated by ultrafiltration. Aliquoted G !  concentrated proteins were snap frozen in liquid N 2  and stored at -80 ¼C. Superdex Gel FiltrationÐР The preferred method of AlF 4-  and MgCl 2  removal was gel filtration of concentrated G !  subunits through Superdex 75 and 200 10/300 GL columns arranged in tandem (GE Healthcare). Superdex chromatography thoroughly removed the chemical, and some minor  protein impurities. G !  subunits (2.5 mg) eluted from the glutathione Sepharose columns with Mg . GDP . AlF 4 were concentrated to 550 " l in CHAPS storage buffer by ultrafiltration. The Superdex columns were equilibrated with CHAPS storage buffer and pre-calibrated with gel filtration sizing standards (BioRad). G !  subunits were  pumped through the columns at 0.3 ml/min using a BioRad Duoflow System and fractions of the column eluate were collected using a fraction collector. Fractions containing monomeric G !  subunits were pooled, concentrated by  b  y g u e  s  t   on J   a n u a r  y 9  ,2  0 1  7 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om    4 ultrafiltration, snap frozen in liquid N 2 , and stored at -80 ¼C. Subcellular FractionationÐР High Five insect cells (25 ml suspension culture) were grown to 2.0 x 10 6  cells/ml in Sf900 II medium (Invitrogen) and infected with 250 " l of twice-amplified G ! i 1 , and GST or GST-Ric-8A baculovirus stocks. Cells were collected by centrifugation and lysed in 12.5 ml of detergent-free buffer (20 mM HEPES-KOH  pH 8.0, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, and protease inhibitor mixture) by nitrogen cavitation using a Parr bomb. Nuclei were removed by centrifugation at 500 x g, and membranes were then separated from soluble  proteins by centrifugation of the 500 x g supernatant at 100,000 x g for 1 h. Reducing Laemmli-sample buffer was added to the supernatant (soluble) and membrane fractions, the samples were boiled and resolved on 10% SDS- polyacrylamide gels containing 4M Urea in the resolving gel. Gels were Western blotted with anti-G ! i 1/2 antiserum (BO84) to detect myristoylated and unmodified G ! i 1  (25). Trypsin protection assaysÐР G !  trypsin  protection assays were performed as described with minor modifications (10, 26-29). G !  subunits (2.5 " M ea) were incubated with 100 " M GDP in HEDL buffer (20 mM HEPES-KOH, pH 8.0, 1 mM EDTA, 0.05% m/v deionized  polyoxyethylene 10 lauryl ether (C12E10) alone, or in HEDL buffer containing 30 " M AlCl 3 , 50 mM MgCl 2 , 10 mM NaF on ice for 30 min. G !  subunits were then incubated for 10 or 30 min. with the following concentrations of trypsin that had been pretreated with 25 ng/ml TPCK; G ! q, 0.1% m/v, (22 ¼C); G ! 13, 0.25% m/v, (22 ¼C); G ! i 1 , 0.25% m/v, (30 ¼C); G ! s s  and G ! olf, 0.5% m/v (30 ¼C). Reactions were quenched by the addition of 40 " g/ml lima bean trypsin inhibitor and reducing SDS-PAGE Laemmli-sample buffer. Samples were boiled, resolved by SDS-PAGE and  protein fragments were visualized by Coomassie- blue staining. GTP  "  S binding assaysÐР Intrinsic and Ric-8-assisted GTP " S binding assays were performed as reported previously (13,30). Purified untagged Ric-8A or Ric-8BFL (200 nM) were mixed with G !  (100 nM) in 20 mM HEPES-KOH, pH 8.0, 100 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 10 mM MgCl 2 , 0.05% m/v Deionized  polyoxyethylene (10) lauryl ether, C12E10 (G ! i 1 , G ! s s , G ! 13) or 0.05% m/v Genapol C-100 (Calbiochem) (G ! q, G ! olf), and 10 " M [ 35 S]-GTP " S (SA 20,000 cpm/pmol). Duplicate aliquots were taken from the reactions at specific time  points, quenched in 20 mM Tris, pH 7.7, 100 mM  NaCl, 10 mM MgCl 2 , 1 mM GTP and 0.08% m/v Deionized polyoxyethylene 10 lauryl ether C12E10, and filtered onto BA-85 nitrocellulose filters (GE Healthcare). The filters were washed with 20 mM Tris pH 7.7, 100 mM NaCl, 2 mM MgCl 2 , dried, and subjected to scintillation counting. To quantify the amount of GTP " S  binding proteins present in the Hi-trap Q G ! q column eluate fractions, 400 nM purified Ric-8A was mixed with each fraction and the assay was  performed for 30 min at 30 ¼C.  Phospholipase C  #   assaysÐР Phospholipid vesicles were prepared as described previously so that the final reaction (60 " l) contained 200 " M  phosphatidylethanolamine (PE) and 50 " M [Inositol-2- 3 H(N)]-phosphatidylinositol 4,5- bisphosphate (PIP 2 ) at 6-8000 cpm/assay (31). PLC # 2 or PLC # 3 were added at 10 ng/assay. G ! q was diluted in buffer containing 20 mM HEPES-KOH, pH 7.2, 100 mM NaCl, 1 mM DTT, 2 mM MgCl 2 , 0.5 mM EDTA, 1 " M GDP and 0.15% m/v # -octylglucoside (final assay concentration). To activate G ! q, G ! q was diluted in the same  buffer but with 10 mM NaF and 30 µM AlCl 3 . The PLC reactions were initiated by addition of 2.8 mM CaCl 2  (1 " M free Ca +2 ) and the samples were incubated at 30 o C. Reactions were terminated by addition of 200 " l 10% m/v trichloroacetic acid, followed by addition of 100 " l of 10 mg/ml BSA. Precipitated proteins and lipids were centrifuged, and 300 " l of the inositol trisphosphate (IP 3 )-containing supernatant was analyzed by liquid scintillation counting. In all assays, blank solutions corresponding to the storage buffers for each of the proteins were included such that all the reactions had exactly the same solution components.   Adenylyl Cyclase assaysÐÐSf  9 cells were infected with recombinant adenylyl cyclase (AC) 3, 5, or 6 baculoviruses. Cells were collected 48 h  post infection, suspended in lysis buffer (20 mM HEPES-KOH, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, 2 mM DTT, protease inhibitor mixture) and lysed by nitrogen cavitation  b  y g u e  s  t   on J   a n u a r  y 9  ,2  0 1  7 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om    5 using a Parr bomb. The cell lysate was centrifuged at 500 x g. The supernatant was centrifuged at 70,000 x g for 30 min to isolate total cell membranes. Membranes were washed, and homogenized into membrane storage buffer (20 mM HEPES-KOH, pH 8.0, 20% m/v sucrose, 1 mM DTT + protease inhibitor mixture) using a dounce homogenizer with tight pestle. Membrane homogenates were frozen and stored at -80 ¼C until use. G proteins were loaded with [ 35 S]-GTP " S and isolated by gel filtration chromatography as described previously (14). Precisely determined G ! -GTP " S concentrations were measured by scintillation counting of a fixed volume of each gel-filtered, monomeric G !  pool. Forskolin and/or G proteins in ATP regeneration  buffer (50 mM HEPES-KOH, pH 8.0, 10 mM MgCl 2 , 10 mM phosphocreatine, 10 units/ml creatine phosphokinase, 10 µ M GTP, 200 µ M ATP, 100 mM 3-isobutyl-1-methylxanthine (IBMX), 100 µ M rolipram) were added to 625 ng membrane homogenate in stimulation buffer (50 mM HEPES-KOH, pH 8.0, 0.05% m/v BSA, 100 µ M IBMX, 100 µ M rolipram) in 96-well format and incubated for 5 min at 22 ¼C. Produced cAMP was detected using a Perkin-Elmer LANCE cAMP detection kit according to the manufacturerÕs instructions and measured in a Victor 3V (Perkin Elmer) plate reader. RESULTS  Ric-8 proteins promote recombinant G !   subunit expression in cellsÐР Genetic ablation of Ric-8 genes in various organisms leads to defects in efficient G !  subunit expression (15-19). Ric-8A  binds all G !  subunits in vitro  except G ! s class and Ric-8B preferentially binds G ! s and G ! q (12,13). We tested whether the expression of G ! s s  or G ! i 1  was up regulated by co-overexpression of Ric-8 homologs. Ric-8A and two Ric-8B isoforms were co-transfected in HEK293 cells with YFP-tagged G ! s s  or G ! i 1  subunits. Fluorescence intensity measurements of intact cells were used (excitation Ð 485 nm, emission Ð 535 nm) to quantify the relative amounts of expressed YFP-G !  in each condition of Ric-8 expression. Ric-8BFL specifically potentiated YFP-G ! s expression, whereas Ric-8A, and to a lesser degree, Ric-8BFL potentiated YFP-G ! i expression (Fig. 1A). These results are consistent with the observed Ric-8 binding specificities to G !  subunits, with the exception that Ric-8B $ 9  binds G ! s, but did not enhance its expression. The insect cell protein expression system is the method of choice for purification of G protein subunits resistant to expression in  E. coli  (8-11). Purification of many insect cell expressed G !  subunits (G ! q-, and G ! 12/13-family) is laborious and results in low yield of final product. We know of no example in which G ! olf has been purified  by this method successfully. Since Ric-8 proteins  promoted G !  subunit expression in mammalian cells, we tested whether they could also potentiate recombinant G !  subunit expression in insect cells for the eventual purpose of using this system to develop an enhanced method of G !  subunit  purification. High-five insect cells were infected with untagged G ! q, or G ! q and GST-Ric-8A recombinant baculoviruses. Whole detergent lysates of pelleted cells were prepared, clarified, and chromatographed over Hi-trap Q anion exchange columns. The columns were washed and eluted with a linear NaCl gradient. Consecutive fractions of the column eluates that contained G ! q were resolved by SDS-PAGE and Coomassie stained, or Western blotted with an anti-G ! q/11 antibody. Co-expression of GST-Ric-8A with G ! q dramatically potentiated the amount of G ! q recovered from the column ~25-fold in comparison to the condition where GST-Ric-8A was not expressed (Fig. 1B). The G ! q obtained from this one-step purification procedure was # 50% pure, and was tested functionally in respect to its capacity to bind GTP " S in a Ric-8A-dependent manner. An equal portion of each Hi-trap Q column eluate fraction was supplemented with purified Ric-8A (400 nM) and allowed to  bind radiolabeled GTP " S for 30 min at 25 ¼C. The amount of active G protein present in each fraction was determined by quantifying the amount of  protein-bound nucleotide using a nitrocellulose filter binding method. The peak G ! q-containing fractions (1 ml ea) from the GST-Ric-8A and G ! q or G ! q alone experiments as judged by the Coomassie gels and Western blots also contained the highest levels of protein-bound GTP " S (4.2 " M and 0.15 " M active G protein respectively) (Fig. 1C). The peak fraction from the GST-Ric-   b  y g u e  s  t   on J   a n u a r  y 9  ,2  0 1  7 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om 
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