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

Biochemical and Biophysical Research Communications
S I ChangS K Namgoong

Abstract

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.

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Citations

Nov 16, 2018·Protein and Peptide Letters·Ghazaleh Arabzadeh, Azar Shahpiri
Jun 25, 2004·The Biochemical Journal·Sun-Mi JungJung-Do Choi
May 21, 1999·Biochemical and Biophysical Research Communications·C K ChongJ D Choi
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Jun 11, 2002·Biochemical and Biophysical Research Communications·Tae-Yeol YoonJung-Do Choi
Jun 25, 2003·Biochemical and Biophysical Research Communications·Dung Tien LeJung-Do Choi

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