title: Expression, purification, and characterization of a human recombinant 17beta-hydroxysteroid dehydrogenase type 1 in Escherichia coli.
abstract: 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.
authors.affiliation: Graduate Institute of Medicine, Kaohsiung Medical University, Taiwan.
authors.forename: Yi-Hsun
authors.initials: YH
authors.lastname: Chang
authors.forename: Yuan-Liang
authors.initials: YL
authors.lastname: Wang
authors.forename: Jain-Yu
authors.initials: JY
authors.lastname: Lin
authors.forename: Lea-Yea
authors.initials: LY
authors.lastname: Chuang
authors.forename: Chi-Ching
authors.initials: CC
authors.lastname: Hwang
mesh_terms: 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*
chemical_list: D011994:Recombinant Proteins; D015067:17-Hydroxysteroid Dehydrogenases; C044256:3 (or 17)-beta-hydroxysteroid dehydrogenase