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Expression of the Arabidopsis G-protein GPα1: purification and characterisation of the recombinant protein

Research paper by Alan Wise, Paul G. Thomas, T. Hedley Carr, Gillian A. Murphy, Paul A. Millner*

Indexed on: 01 Mar '97Published on: 01 Mar '97Published in: Plant Molecular Biology



Abstract

The Arabidopsis Gα subunit, GPα1, was expressedwithin Escherichia coli by co-transformation with the expressionvector and the dnaY gene which encodes tRNAArgAGA/AGG. Isolation of the recombinant GPα1 in a highly pureform could be achieved by a combination of anion exchange and dyeaffinity chromatography or by a single step affinity procedure viachromatography on 4-amino-anilido-GTP agarose. The recombinant proteinyielded by both procedures was highly active and bound GTPγS withan apparent Kd in the nM range. GTPγS binding wasstimulated two-fold in the presence of Zn2+ compared with that inthe presence of Mg2+, Mn2+ or Ca2+.Abbreviations: 4aaGTP, 4-amino-anilido-GTP; GTPγS,guanosine- 5′-(3-O-thiotriphosphate), PMSF,phenylmethylsulphonyl fluoride; PVDF, polvinylidene fluoride;rGPα1, recombinant GPα1