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

Expression of the Arabidopsis G-protein GPα1: purification and characterisation of the... The Arabidopsis Gα subunit, GPα1, was expressedwithin Escherichia coli by co-transformation with the expressionvector and the dnaY gene which encodes tRNAArg AGA/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 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Molecular Biology Springer Journals

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

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Publisher
Kluwer Academic Publishers
Copyright
Copyright © 1997 by Kluwer Academic Publishers
Subject
Life Sciences; Biochemistry, general; Plant Sciences; Plant Pathology
ISSN
0167-4412
eISSN
1573-5028
D.O.I.
10.1023/A:1005732423622
Publisher site
See Article on Publisher Site

Abstract

The Arabidopsis Gα subunit, GPα1, was expressedwithin Escherichia coli by co-transformation with the expressionvector and the dnaY gene which encodes tRNAArg AGA/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

Journal

Plant Molecular BiologySpringer Journals

Published: Sep 29, 2004

References

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