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.
Chang
S I
SI
Department of Biochemistry, Chungbuk National University, Cheongju, Korea. sichang@cbucc.chungbuk.ac.kr
Kang
M K
MK
Choi
J D
JD
Namgoong
S K
SK
D000095:Acetolactate Synthase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006540:Herbicides / Q000378:metabolism; D007700:Kinetics; D010947:Plants, Toxic; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D014026:Nicotiana / Q000201:enzymology*
D006540:Herbicides; D011994:Recombinant Proteins; D000095:Acetolactate Synthase