Functional expression of Arabidopsis thaliana anthranilate synthase subunit I in Escherichia coli

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 μM), and the feedback inhibition by Trp (complete inhibition by 10 μM 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 sitedirected mutagenesis modifications of the Trp allosteric site, inactivated the recombinant protein.