AI Article Synopsis
- The human enzyme 3beta-hydroxysteroid dehydrogenase type 1 (3beta-HSD1) was overexpressed and purified using a baculovirus system and nickel-affinity chromatography, achieving over 95% purity.
- The purification process involved solubilizing the membrane protein with the detergent C(12)E(8) and yielded about 3-4 mg of active enzyme from a significant number of infected cells.
- Kinetic analysis indicated the enzyme has a K(m) of 1.7 microM and V(max) of 50 nmol/min/mg, laying groundwork for further studies on its structure and function.
Article Abstract
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.
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| http://dx.doi.org/10.1006/prep.1999.1180 | DOI Listing |
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