Study on Protein-protein Interaction of the Key Enzyme 3 ,17β-HSD for Steroid Degradation by Comamonas Testosteroni
In order to understand protein-protein interaction of the key enzyme 3 ,17β-HSD for steroid degradation in Comamonas testosteroni (C. testosteroni) ATCC11996 ,reveal steroid degradation mechanism in the future. Constructed 3 ,17β-HSD gene knockout mutant byhomologous recombination and frameshift mutation. Under the condition of 95％testosterone recovery rate ,detected testosterone degradation by wild-type and 3 ,17β-HSD gene mutantC. testosteronis-trains with HPLC. The ability for steroid degradationof mutant strain was significantly lower than the wild-type strain. Expression and purification 3,17β-HSD in E.coli system to obtain purified protein,the concentration was 2.4 mg/mL. Preparation specific mouse polyclonal antibody with 3 ,17β-HSD protein ,ELISA detected antibody titer was 1:320000. Co-immunoprecipitation (Co-IP) was performed with 3 ,17β-HSD polyclonal antibody and protein complexes from cell lysates of testosterone induced wild type C. testosteroni. Detected protein-protein interaction with mass spectrometer. The results showed that short chain dehydrogenase SDRy(WP-003078287.1) was the major interacting protein of 3 ,17β-HSD for testosterone degradation in C. testosteroni.
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