Baculovirus expression of mammalian G protein α subunits

FEBS Letters

Torossian, Krikor¹  Dennis, Michael¹  Caron, Mireille¹  Labrecque, Jean¹  Plamondon, Josée¹ 

[1] Biotechnology Research Institute, 6100 Royalmount Ave., Montréal, QC, Canada

关键词: GTP-binding protein;    Heterologous expression;    Baculovirus;    GTP-γ-S;    guanosine 5′-γ-thiotriphosphate;    GTP;    guanosine triphosphate;    GDP;    guanosine diphosphate;    ATP;    adenosine triphosphate;    PBS;    phosphate-buffered isotonic saline;    SDS-PAGE;    sodium dodecyl sulfate polyacrylamide gel electrophoresis;    AcNPV;    Autographa californica nuclear polyhedrosis virus;   

DOI  :  10.1016/0014-5793(92)80609-K

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Complementary DNAs encoding three subtypes of the α subunit (α_i−1, αo 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 [³⁵S]methionine or [³H]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-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.

【 授权许可】

Unknown