Purification of recombinant G proteins from Sf9 cells by hexahistidine tagging of associated subunits: Characterization of α₁₂ and inhibition of adenylyl cyclase by α_z

A method is described for purification of G protein α and βγ subunits from Sf9 cells infected with recombinant baculoviruses. The subunit to be purified is coexpressed with an associated subunit bearing a hexahistidine tag. After adsorption of the oligomer to a Ni²⁺-containing column, the subunit to be purified is eluted specifically by promoting subunit dissociation with AlF^-₄. The α subunits of G₁₂, G_q, G_z, and G_i1 and the β₁γ₂ subunit complex were easily and efficiently purified by this method. Results were superior to established procedures in all cases. Purified α₁₂ was characterized for the first time. The protein has a slow rate of guanine nucleotide exchange (k_on,GTPγS = 0.01 min^-1) and a very slow k_cat for hydrolysis of GTP (0.1-0.2 min^-1). GTPγS (guanosine 5′-3-O-(thio)triphosphate)·α₁₂ does not influence the activity of several adenylyl cyclases or phospholipases. Activated α_z inhibits the activity of type I and type V adenylyl cyclases. It is a somewhat more potent inhibitor of type V adenylyl cyclase than is activated α_i1.