Complementary DNAs encoding three subtypes of the α subunit (αi−1, αo and αx) 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 α chains, and co-migrated with the native proteins from rat brain membranes in immunoblotting analyses. Soluble and particulate forms of all three immunoreactive α chains were observed following ultracentrifugation of cell lysates. Biosynthetic radiolabelling of infected cells with [35S]methionine or [3H]myristate showed that both soluble and particulate forms of αi−1 and αo were myristoylated; in contrast, α, did not incorporate myristate. The soluble fractions from cells expressing α chains showed high levels of GTP-binding activity over that observed in uninfected cells, or in cells infected with wild-type virus. The peak expression levels observed at 72 h post-infection were highest for αo at ca, 400 pmol of GTP-γ-35S/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 α chains which retain GTP-binding activity and are appropriately modified by myristoylation.
