Purification of Heterotrimeric G Protein α Subunits by GST-Ric-8 Association

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 Gαolf for the first time and unprecedented yield of Gαq and Gα13. 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-AlF4− (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 Gαolf showed that it binds GTPγS at a rate marginally slower than Gαs short and directly activates adenylyl cyclase isoforms 3, 5, and 6 with less efficacy than Gαs short.