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

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Abstract
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.