Low temperature cultivation of Escherichia coli carrying a rice lipoxygenase L-2 cDNA produces a soluble and active enzyme at a high level.
Using a T7 RNA polymerase promoter system, rice lipoxygenase L-2 cDNA was expressed in E. coli as a fusion protein with 18 amino acid residues at the amino terminal end of the original enzyme. Incubation at 37 degrees C for 3 h in the presence of the inducer resulted in the production of inactive lipoxygenase. However, when induction was carried out at 15 degrees C for 16 h, active lipoxygenase, amounting to 3% of the total soluble protein, was produced. The enzyme was purified by ammonium sulfate precipitation and Mono-Q column chromatography to homogeneity at a yield of 80%. Expression of this protein should permit future site-directed mutagenesis of the gene and crystallization of the enzyme.
Shirano
Y
Y
Mitsui Plant Biotechnology Research Institute, Tsukuba, Ibaraki, Japan.
Shibata
D
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000096:biosynthesis*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D008084:Lipoxygenase / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology*; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D012995:Solubility; D013696:Temperature; D014764:Viral Proteins
D004274:DNA, Recombinant; D014764:Viral Proteins; C066223:lipoxygenase L-2, rice; D008084:Lipoxygenase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases