FUNCTIONAL EXPRESSION OF ARABIDOPSIS-THALIANA ANTHRANILATE SYNTHASE SUBUNIT-I IN ESCHERICHIA-COLI

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 V-max (45 nmol min(-1) mg(-1)), the apparent KM for chorismate (180 mu M), and the feedback inhibition by Trp (complete inhibition by 10 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.