Molecular cloning,expression and characterization of the human recombinant 17β-hydroxysteroid dehydrogenase type 1 in E.coli

• Main Authors: Jain-Yu Lin, 林建宇
• Other Authors: Chi-Ching Hwang
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/78869863979430817954

碩士 === 高雄醫學大學 === 生物化學研究所 === 96 === Human 17β-hydroxysteroid dehydrogenases 1(17β-HSD1) catalyzes the conversion of an inactive estrone to the biologically active 17β-estradiol. It plays an important role in the biosynthesis as well as metabolism of steroid hormones, and may involve in the development of hormone-dependent breast cancer. In this study we have cloned, overexpressed and purified the 17β-HSD1 in E.coli. The highly expressed 17β-HSD1 results in the formation of inclusion body and a minor soluble protein. The enzyme uses either NADP+ or NAD+ as a cofactor with prefered for NADP+ in the oxidation of the hydroxysteroid. Furthermore, an initial velocity pattern obtained by varying the 17β-estradiol concentrations at different fixed concentrations of NAD(P)+ at pH 7.4 shows an intersection on the left of the ordinate in a double reciprocal plot, suggesting a sequential kinetic mechanism of 17β-HSD1 catalyzed the reaction. The inclusion body can be dissolved in 6M urea solution, while the activity is not recovery after refolding through dialysis. There is a difference in circular dichroism spectrum between refolded protein and soluble proteins. This suggests the loss of activity may due to an improper folding. The protein is stable in neutral environment (pH7.4). Protein aggregates in acid environment (pH5.8), and causes the decrease in activity. No activity was observed in basic condition (pH11). Although tried to express 17β-HSD1 in yeast system , we can’t detect 17β-HSD1.