BACULOVIRUS EXPRESSION OF MAMMALIAN G-PROTEIN ALPHA SUBUNITS

Complementary DNAs encoding three subtypes of the alpha-subunit (alpha(i-1), alpha(o) and alpha(s)) of rat guanyl nucleotide regulatory proteins were used to construct recombinant baculoviruses which direct high-level expression of the corresponding proteins in cultured Sf9 insect cells. The expressed proteins were recognized by polyclonal antisera specific for the different alpha-chains, and co-migrated with the native proteins from rat brain membranes in immunoblotting analyses. Soluble and particulate forms of all three immunoreactive alpha-chains were observed following ultracentrifugation of cell lysates. Biosynthetic radiolabelling of infected cells with [S-35]methionine or [H-3]myristate showed that both soluble and particulate forms of alpha(i-1) and alpha(o) were myristoylated; in contrast, alpha(s) did not incorporate myristate. The soluble fractions from cells expressing alpha-chains showed high levels of GTP-binding activity over that observed in unifected cells, or in cells infected with wild-type virus. The peak expression levels observed at 72 h post-infection were highest for alpha(o) at ca. 400 pmol of GTP-gamma-S-35/mg protein, or roughly 2% of the total soluble protein. The results of this work show that the baculovirus system can be employed for high-level production of mammalian G protein alpha-chains which retain GTP-binding activity and are appropriately modified by myristoylation.