Soluble overexpression in Escherichia coli, and purification and characterization of wild-type recombinant tobacco acetolactate synthase

Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of L-leucine, L-isoleucine, and L-valine. The wild-type ALS gene from Nicotiana tabacum was cloned into the bacterial expression vector pGEX-2T. The resulting recombinant plasmid pGEX-ALS2 was used to transform Escherichia coli strain XL1-Blue, and the wild-type tobacco ALS (wALS) was expressed in the bacteria as a protein fused with glutathione S-transferase (GST). The fusion product GST-wALS was purified in a single step on a glutathione-Sepharose column. The purified GST-wALS was sensitive to a sulfonylurea herbicide, and was lost its sensitivity to end products, L-valine, L-leucine and L-isoleucine. These results suggest that the purified recombinant tobacco ALS was functionally active, and that the sulfonylureas may not bind to the feedback regulatory site on the plant ALS. (C) 1997 Academic Press.