The Optimization Of Expression, Purification, And Storage Of3αHydroxysteroid Dchydrogenase

3a-Hydroxysteroid Dehydrogenase (3a-HSD) is a steroid dehydrogenase created by testosterone microcyclus. It can effect various steroid groundsubstance and catalize oxidation-reduction of3α-hydroxy of C19～27steroid/ketone group adversely. Bile acid is one of substrates of3α-HSD.3α-HSD is used as tool enzyme to determine the concentration of total bile acids (TBA) in human serum, and to test the liver function clinically. At the moment, the tool enzymes used for TBA determination are mostly extracted from testosterone pseudomonad directly.The extracting technology is complicated and the procedure is elaborate, so the price of natural3α-HSD separated from bacteria directly is very high. This restricts the generalization of this enzyme on Clinical test.The expressing clone pThioHisA-HSD for HSD was constructed by PCR and plasmid construction. The bacteria strains used for expression were carefully screened, to select a higher expression clone. The induction time,induction temperature, and the concentration of IPTG (Isopropyl (3-D-Thiogalactoside) were optimized. So we determined the expression condition for HSD, that is inducing the recombinant strain with1mM IPTG at the37degree for6hours.When ammonium sulfate concentration was50-60%, most HSD was precipitated down, and most impurity protein was get off. By comparing the effct of different chromatography purification conditions, we find both the Gel Filttration(GF) and ion exchange chromatography(IEX) can separate the HSD effectly. But the activity of HSD recover better with IEX than GF. We also optimized the storage condition of enzyme, the temperature, pH and other conditions were compared and optimized.,and find3a-HSD is stable under the conditions of less than25degree and pH7.4. The study provides valuable consult for decrease the spending of liver function test.