Ric-8A and Ric-8B are nonreceptor 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-8⅐G␣ 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 guanosine 5-[␥-thio]triphosphate (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 short and directly activates adenylyl cyclase isoforms 3, 5, and 6 with less efficacy than G␣ s short .Heterotrimeric G proteins are the foremost signal-transducing molecules used by G protein-coupled-receptors (GPCRs) 3 to regulate sensation and cellular physiology. Agonist-stimulated GPCRs are guanine nucleotide exchange factors that stimulate G protein ␣ subunit (G␣) GDP release. Subsequent GTP binding to G␣ causes heterotrimer dissociation or rearrangement 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 nontraditional 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 Escherichia 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 eluting 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 (Յ50 -200 g of protein/liter of cell culture) (8 -11). To ...