Graduate Institute of Medicine, Kaohsiung Medical University, Taiwan.
Mol Biotechnol. 2010 Feb;44(2):133-9. doi: 10.1007/s12033-009-9221-5.
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.
人 17β-羟类固醇脱氢酶 1 型（17β-HSD1）催化雌酮与 NADPH 的反应，形成雌二醇和 NADP（+），从而调节各种组织中甾体性激素的生物活性。在这里，我们提出了一种从大肠杆菌中高效表达和纯化人 17β-HSD1 的方法。构建了表达载体 pET28a/17β-HSD1 并转化入大肠杆菌 BL21（DE3）细胞。我们发现，通过在 0.25mM IPTG、13°C 下诱导 17β-HSD1 表达过夜，可以获得活性酶。该蛋白通过一步 Ni-NTA 亲和层析进行纯化，从 2.8mg/L 的培养物中获得 2.8mg/L 的产量。动力学研究表明，在 pH9.3 下，以 NADP（+）为辅助因子氧化雌二醇时，V/E（t）为（1.21+/-0.05）x10（-2）/s，K（雌二醇）为 0.8μM。用于重组 17β-HSD1 的新型细菌表达系统可用于大量易纯化，并将有助于该酶的功能研究。
