Low temperature cultivation of Escherichia coli carrying a rice lipoxygenase L‐2 cDNA produces a soluble and active enzyme at a high level

FEBS Letters

Shibata, Daisuke¹  Shirano, Yumiko¹ 

[1] Mitsui Plant Biotechnology Research Institute, Tsukuba, Ibaraki, Japan

关键词: Lipoxygenase;    Inclusion body;    Low temperature cultivation of E. coli;    T7 RNA polymerase promoter;    IPTG;    isopropyl-β-D-thiogalactopyranoside;    SDS;    sodium dodecyl sulfate;   

DOI  :  10.1016/0014-5793(90)80388-Y

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Using at 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°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°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.

【 授权许可】

Unknown