Purification of heterotrimeric G protein alpha subunits by GST-Ric-8 association: primary characterization of purified G alpha(olf) |
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Authors: | Chan PuiYee Gabay Meital Wright Forrest A Kan Wei Oner Sukru S Lanier Stephen M Smrcka Alan V Blumer Joe B Tall Gregory G |
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Affiliation: | Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642, USA. |
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Abstract: | Ric-8A and Ric-8B are nonreceptor G protein guanine nucleotide exchange factors that collectively bind the four subfamilies of G protein α subunits. Co-expression of Gα subunits with Ric-8A or Ric-8B in HEK293 cells or insect cells greatly promoted Gα protein expression. We exploited these characteristics of Ric-8 proteins to develop a simplified method for recombinant G protein α subunit purification that was applicable to all Gα subunit classes. The method allowed production of the olfactory adenylyl cyclase stimulatory protein Gα(olf) for the first time and unprecedented yield of Gα(q) and Gα(13). Gα subunits were co-expressed with GST-tagged Ric-8A or Ric-8B in insect cells. GST-Ric-8·Gα complexes were isolated from whole cell detergent lysates with glutathione-Sepharose. Gα subunits were dissociated from GST-Ric-8 with GDP-AlF(4)(-) (GTP mimicry) and found to be >80% pure, bind guanosine 5'-[γ-thio]triphosphate (GTPγS), and stimulate appropriate G protein effector enzymes. A primary characterization of Gα(olf) showed that it binds GTPγS at a rate marginally slower than Gα(s short) and directly activates adenylyl cyclase isoforms 3, 5, and 6 with less efficacy than Gα(s short). |
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Keywords: | Adenylate Cyclase (Adenylyl Cyclase) G Proteins Phospholipase C Protein Purification Signal Transduction Ric-8 Guanine Nucleotide Exchange Factor Myristoylation |
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