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 biosynth esis 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 tran sform Escherichia coli strain XL1-Blue, and the wild-type tobacco ALS (wALS) was expressed in the bacteria as a protein fused with glutathio ne S-transferase (GST). The fusion product GST-wALS was purified in a single step on a glutathione-Sepharose column. The purified GST-wALS w as 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 act ive, and that the sulfonylureas may not bind to the feedback regulator y site on the plant ALS. (C) 1997 Academic Press.