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PLANT PHYSIOLOGY , Vol 106, Issue 1 353-358, Copyright © 1994 by American Society of Plant Biologists

METABOLISM AND ENZYMOLOGY

Functional Expression of Arabidopsis thaliana Anthranilate Synthase Subunit I in Escherichia coli

P. Bernasconi, E. W. Walters, A. R. Woodworth, D. L. Siehl, T. E. Stone and M. V. Subramanian
Sandoz Agro Inc., Research Division, 975 California Avenue, Palo Alto, California 94304-1104

Anthranilate synthase is involved in tryptophan (Trp) biosynthesis. Functional expression of subunit I from Arabidopsis (ASA1) was achieved in bacteria as a protein fused with glutathione S-transferase (GST). The active product was purified in a single step on a glutathione-Sepharose column. The Vmax (45 nmol min-1 mg-1), the apparent KM for chorismate (180 [mu]M), and the feedback inhibition by Trp (complete inhibition by10 [mu]M Trp) of the purified fusion product (GST-ASA1) were comparable to anthranilate synthase purified from plants. Polyclonal antibodies raised against the fusion protein product and purified by affinity chromatography on a GST-ASA1-Sepharose column cross-reacted with a 61.5-kD protein in a partially purified anthranilate synthase preparation from corn seedlings. GST-ASA1 cleavage by thrombin, as well as site-directed mutagenesis modifications of the Trp allosteric site, inactivated the recombinant protein.


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