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

Ric-8A and Ric-8B are nonreceptor G protein guanine nucleotide exchange factors that collectively bind the four subfamilies of G protein alpha subunits. Co-expression of G alpha subunits with Ric-8A or Ric-8B in HEK293 cells or insect cells greatly promoted G alpha protein expression. We exploited these characteristics of Ric-8 proteins to develop a simplified method for recombinant G protein alpha subunit purification that was applicable to all G alpha subunit classes. The method allowed production of the olfactory adenylyl cyclase stimulatory protein G alpha(olf) for the first time and unprecedented yield of G alpha(q) and G alpha(13). G alpha subunits were co-expressed with GST-tagged Ric-8A or Ric-8B in insect cells. GST-Ric-8-G alpha complexes were isolated from whole cell detergent lysates with glutathione-Sepharose. G alpha subunits were dissociated from GST-Ric-8 with GDP-AlF4- (GTP mimicry) and found to be > 80% pure, bind guanosine 5 '-[gamma -thio]triphosphate (GTP gamma S), and stimulate appropriate G protein effector enzymes. A primary characterization of G alpha(olf) showed that it binds GTP gamma S at a rate marginally slower than G alpha(s short) and directly activates adenylyl cyclase isoforms 3, 5, and 6 with less efficacy than G alpha(s short).