Expression, characterization and purification of soluble G‐protein βγ dimers composed of defined subunits in baculovirus‐infected insect cells

Abstract

Recombinant β₁γ₂ dimers of signal-transducing guanine nucleotide-binding proteins (G-proteins) carrying a mutation known to block isoprenylation of the γ₂ subunit were expressed as a soluble protein in baculovirus-infected insect cells. The soluble βγ dimer was analyzed by sucrose density gradient centrifugation and purified to near homogeneity in the absence of detergents. The sedimentation velocity studies gave an s _20,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 βγ dimers suitable for functional and structural characterization.