Membrane-bound human 3 beta-hydroxysteroid dehydrogenase: Overexpression with His-tag using a Baculovirus system and single-step purification

The membrane-bound human 3 beta-hydroxysteroid dehydrogenase type 1 (3 beta -HSD1) was overexpressed with His(6)-tag, using a baculovirus expression sy stem, and then purified by nickel-chelated affinity chromatography. Overexp ression of 3 beta-HSD1 was confirmed by enzyme assay and Western blot analy sis. The protein was purified to more than 95% homogeneity by a single-step Ni2+-chelated affinity chromatography after solubilization of the membrane -bound protein with the detergent C12E8. High yield was repeatedly obtained , with 3-4 mg of homogeneous and active 3 beta-HSD1 from 1 x 10(9) of infec ted Sf9 cells. The kinetic study showed a K-m of 1.7 mu M and a V-max of 50 nmol/min/mg of purified protein using dehydroepiandrosterone as the substr ate. The above preparation will facilitate the structure-function study of this important enzyme. (C) 2000 Academic Press.