Expression of the Arabidopsis G-protein GP alpha 1: purification and characterisation of the recombinant protein

The Arabidopsis Galpha subunit, GPalpha1, 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...

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Bibliographic Details
Published in:Plant molecular biology 1997-03, Vol.33 (4), p.723-728
Main Authors: Wise, A, Thomas, P.G, Carr, T.H, Murphy, G.A, Millner, P.A
Format: Article
Language:English
Subjects:
Arabidopsis
Arabidopsis thaliana
binding
binding proteins
calcium
dnay gene
Escherichia coli
gene transfer
genes
genetic transformation
guanosine triphosphate
inorganic ions
magnesium
manganese
metal ions
plant proteins
recombinant proteins
transfer RNA
zinc
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Summary:The Arabidopsis Galpha subunit, GPalpha1, 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 GTP gammaS with an apparent Kd in the nM range. GTP gammaS binding was stimulated two-fold in the presence of Zn2+ compared with that in the presence of Mg2+, Mn2+ or Ca2+.
ISSN:0167-4412
1573-5028
DOI:10.1023/A:1005732423622