Effect of ethanol and low-temperature culture on expression of soybean lipoxygenase L-1 in Escherichia coli.
We have constructed a full-length cDNA that encodes soybean seed lipoxygenase L-1 and have expressed it in Escherichia coli. This gene was inserted into a pT7-7 expression vector, containing the T7 RNA polymerase promoter. E. coli, strain BL21 (DE3), which carries the T7 promoter in its genome, was transfected with the plasmid. Expression of this gene when the cells were cultured at 37 degrees C yielded polypeptide that was recognized by anti-L-1 antibody, but had very little lipoxygenase activity. Yields of active enzyme were markedly increased when cells were cultured at 15-20 degrees C. When ethanol, which has been reported to be an excellent elicitor of heat-shock proteins in E. coli, was also present at a level of 3% the yield was further increased by 40%. Under optimum conditions 22-30 mg of soluble active enzyme was obtained per liter of culture.
Steczko
J
J
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153.
Donoho
G A
GA
Dixon
J E
JE
Sugimoto
T
T
Axelrod
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003080:Cold Temperature; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D000431:Ethanol / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D008084:Lipoxygenase / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology* / Q000235:genetics*; D010957:Plasmids; D013025:Glycine max / Q000201:enzymology / Q000235:genetics
D000431:Ethanol; D004247:DNA; D008084:Lipoxygenase