Huang Y W, Lu M L, Qi H, Lin S X
Medical Research Council Group in Molecular Endocrinology, CHUL Research Center and Laval University, Quebec, Quebec, G1V 4G2, Canada.
Protein Expr Purif. 2000 Mar;18(2):169-74. doi: 10.1006/prep.1999.1180.
The membrane-bound human 3beta-hydroxysteroid dehydrogenase type 1 (3beta-HSD1) was overexpressed with His(6)-tag, using a baculovirus expression system, and then purified by nickel-chelated affinity chromatography. Overexpression of 3beta-HSD1 was confirmed by enzyme assay and Western blot analysis. The protein was purified to more than 95% homogeneity by a single-step Ni(2+)-chelated affinity chromatography after solubilization of the membrane-bound protein with the detergent C(12)E(8). High yield was repeatedly obtained, with 3-4 mg of homogeneous and active 3beta-HSD1 from 1 x 10(9) of infected Sf9 cells. The kinetic study showed a K(m) of 1.7 microM and a V(max) of 50 nmol/min/mg of purified protein using dehydroepiandrosterone as the substrate. The above preparation will facilitate the structure-function study of this important enzyme.
