EXPRESSION, CHARACTERIZATION AND PURIFICATION OF SOLUBLE G-PROTEIN BETA-GAMMA DIMERS COMPOSED OF DEFINED SUBUNITS IN BACULOVIRUS-INFECTED INSECT CELLS

Recombinant beta1gamma2 dimers of signal-transducing guanine nucleotide-binding proteins (G-proteins) carrying a mutation known to block isoprenylation of the gamma2 subunit were expressed as a soluble protein in baculovirus-infected insect cells. The soluble betagamma dimer was analyzed by sucrose density gradient centrifugation and purified to near homogeneity in the absence of detergents. The sedimentation velocity studies gave an s20,w value of 4.1 +/- 0.4 S. The two subunits segregated as a dimer upon sucrose density gradient centrifugation and purification by sequential ion exchange and hydroxylapatite chromatography. The results show that baculovirus-infected insect cells can be employed for high level production of pure G-protein betagamma dimers suitable for functional and structural characterization.