期刊
MOLECULAR BIOTECHNOLOGY
卷 44, 期 2, 页码 133-139出版社
HUMANA PRESS INC
DOI: 10.1007/s12033-009-9221-5
关键词
17 beta-Hydroxysteroid dehydrogenase 1; Short chain dehydrogenase/reductase; Protein purification; Protein expression; Enzyme catalysis
资金
- National Science Council (NSC) [NSC 97-2320-B-037-043, NSC 96-2320-B-037-063]
Human 17 beta-hydroxysteroid dehydrogenase type 1 (17 beta-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 17 beta-HSD1 from Escherichia coli. The expression vector pET28a/17 beta-HSD1 was constructed and transformed into Escherichia coli BL21(DE3) cells. We found that the active enzyme can be obtained by inducing 17 beta-HSD1 expression at 0.25 mM IPTG, 13A 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 +/- A 0.05) x 10(-2)/s and K (estradiol) of 0.8 mu M in the oxidation of estradiol with NADP(+) as cofactor at pH 9.3. The new bacterial expression system for recombinant 17 beta-HSD1 is useful for the easy purification of large amounts and will facilitate the functional study of this enzyme.
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