Premium
Baculovirus expression of mammalian G protein α subunits
Author(s) -
Labrecque Jean,
Caron Mireille,
Torossian Krikor,
Plamondon Josée,
Dennis Michael
Publication year - 1992
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(92)80609-k
Subject(s) - sf9 , myristoylation , gtp' , g protein , biochemistry , myristic acid , protein subunit , recombinant dna , microbiology and biotechnology , ultracentrifuge , antiserum , polyclonal antibodies , biology , methionine , chemistry , amino acid , membrane , gene , antibody , enzyme , fatty acid , spodoptera , receptor , palmitic acid , immunology
Complementary DNAs encoding three subtypes of the α subunit (α i−1 , α o and α x ) 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 [ 35 S]methionine or [ 3 H]myristate showed that both soluble and particulate forms of α i−1 and α o 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 α o at ca, 400 pmol of GTP‐γ‐ 35 S/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.