Expression and purification of functional G protein alpha subunits using a baculovirus expression system.
The alpha subunits of the heterotrimeric guanine nucleotide-binding proteins Gi1, Gi2, Gi3, G0, and Gs have been overexpressed in Sf9 cells using a baculovirus expression system. The Gi1 alpha, Gi2 alpha, Gi3 alpha, and G0 alpha have been purified to homogeneity from infected Spodoptera frugiperda (SF9) cells and characterized. Yields of up to 1.8 mg of purified recombinant G alpha have been obtained from 300-ml cultures of infected cells. The recombinant alpha subunits are myristoylated and are ADP-ribosylated by pertussis toxin only in the presence of beta gamma subunits. They bind guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) with low nM dissociation constants and stoichiometries of 0.8 mol/mol or greater. The rGi1 alpha, rGi2 alpha, and rGi3 alpha are capable of interacting with angiotensin II receptors based on their ability to restore high affinity angiotensin II binding in rat liver membranes shifted to a low affinity state with GTP gamma S.
Graber
S G
SG
Department of Pharmacology, University of Virginia School of Medicine, Charlottesville 22908.
Figler
R A
RA
Garrison
J C
JC
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D037342:Pertussis Toxin; D051381:Rats; D011945:Receptors, Angiotensin / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D011945:Receptors, Angiotensin; D010566:Virulence Factors, Bordetella; D000804:Angiotensin II; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins