Baculovirus expression and purification of a soluble, mutant G-protein alpha subunit.
The cDNA for the alpha i1 protein that had undergone site-directed mutagenesis to change glycine-2 to alanine was ligated into a baculovirus transfer vector. A recombinant virus was obtained by transfecting Sf9 cells with both the wild-type baculovirus DNA and the transfer vector and screening for recombinant plaques. Infection with the recombinant virus led to a high level of expression of the mutated alpha i1 protein in the soluble fraction of the cell. The protein was purified by ammonium sulfate precipitation, gel filtration, and immobilized dye chromatography. The typical yield was 7.5 mg from two 800-ml cultures. The protein showed immunoreactivity to three different alpha i-specific antibodies. It bound guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) with a stoichiometry of 0.63 to 0.91 mol/mol and with a rate constant (kGTP gamma S) of binding of 0.126 min-1. When GTP gamma S bound, the protein was protected from complete tryptic cleavage. The recombinant protein was able to undergo pertussis toxin-catalyzed ADP ribosylation and bind beta gamma subunits but with a reduced affinity compared to that of alpha transducin. Thus using a recombinant baculovirus, a nonmyristylated G protein alpha subunit was abundantly expressed in Sf9 cells and milligram quantities of a functional protein were easily purified.
Jones
T L
TL
Molecular Pathophysiology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Woodard
C
C
Spiegel
A M
AM
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007313:Insecta; D008969:Molecular Sequence Data; D009154:Mutation; D051381:Rats
D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004247:DNA; D019204:GTP-Binding Proteins