Expression of the Arabidopsis G-protein GP alpha1: purification and characterisation of the recombinant protein.
The Arabidopsis G alpha subunit, GP alpha1, was expressed within Escherichia coli by co-transformation with the expression vector and the dnaY gene which encodes tRNA(Arg)(AGA/AGG) Isolation of the recombinant GP alpha1 in a highly pure form could be achieved by a combination of anion exchange and dye affinity chromatography or by a single step affinity procedure via chromatography on 4-amino-anilido-GTP agarose. The recombinant protein yielded by both procedures was highly active and bound GTPgammaS with an apparent Kd in the nM range. GTPgammaS binding was stimulated two-fold in the presence of Zn2+ compared with that in the presence of Mg2+, Mn2+ or Ca2+.
Wise
A
A
Department of Biochemistry and Molecular Biology, University of Leeds, UK.
Thomas
P G
PG
Carr
T H
TH
Murphy
G A
GA
Millner
P A
PA
D017360:Arabidopsis / Q000235:genetics; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012347:RNA, Transfer, Arg / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D010940:Plant Proteins; D012347:RNA, Transfer, Arg; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins