Expression, purification, and characterization of a human recombinant 17beta-hydroxysteroid dehydrogenase type 1 in Escherichia coli.
Human 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) catalyzes the reaction of estrone with NADPH to form estradiol and NADP(+), thereby regulating the biological activity of sex steroid hormones in a variety of tissues. Here, we present an efficient method for expressing and purifying human 17beta-HSD1 from Escherichia coli. The expression vector pET28a/17beta-HSD1 was constructed and transformed into Escherichia coli BL21(DE3) cells. We found that the active enzyme can be obtained by inducing 17beta-HSD1 expression at 0.25 mM IPTG, 13 degrees C for overnight. The protein is purified by single step Ni-NTA affinity chromatography and yields 2.8 mg/L of culture. The kinetic study shows V/E ( t ) of (1.21 +/- 0.05) x 10(-2)/s and K (estradiol) of 0.8 microM in the oxidation of estradiol with NADP(+) as cofactor at pH 9.3. The new bacterial expression system for recombinant 17beta-HSD1 is useful for the easy purification of large amounts and will facilitate the functional study of this enzyme.
Chang
Yi-Hsun
YH
Graduate Institute of Medicine, Kaohsiung Medical University, Taiwan.
Wang
Yuan-Liang
YL
Lin
Jain-Yu
JY
Chuang
Lea-Yea
LY
Hwang
Chi-Ching
CC
D015067:17-Hydroxysteroid Dehydrogenases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D011994:Recombinant Proteins; D015067:17-Hydroxysteroid Dehydrogenases; C044256:3 (or 17)-beta-hydroxysteroid dehydrogenase