Study On The Protein Interaction Between Hormone-degrading Enzyme 3,17?-HSD And 3?-HSD In Comamonas Testosteroni

Comamonas testosteroni(C.T.)is a kind of bacteria that can degrade steroid hormones.Many proteins are involved in the process of hormone degradation,and proteins rarely play a role alone.The bioremediation of testosterone clumps and the mechanism and expression regulation of hormone degradation-related genes have become a research hotspot.3,17?-HSD and 3?-HSD are two important hormone-degrading enzymes in Phaeomonas testosterone ATCC11996,and SDRy is similar to the up-regulated level of these two enzymes in testosterone-induced transcriptome analysis of C.T bacteria,and some studies in our laboratory have proved that they are also involved in the hormone degradation of C.T.bacteria.By means of gene knockout,recombinant plasmid construction,high performance liquid chromatography,enzyme-linked immunosorbent assay,co-immunoprecipitation and plasmid cotransformation,the protein interaction group between 3,17?-HSD and 3?-HSD in C.T strain was studied.The gene knockout strains of 3,17?-HSD and 3?-HSD were co-nstructed,and the hormone degradation ability of the knockout strains was detected by HPLC.The proteins of 3,17?-HSD and 3?-HSD were prokaryotic expressed and purified,and the polyclonal antibodies of the two proteins were prepared.The obtained antibodies were immunoprecipitated with the whole protein of C.T induced by testosterone,and the interacting proteins were preliminarily analyzed.3,17?-HSD antibody was used for immunoprecipitation and glue strip identification.The co-transformation experiment was carried out and the effect of co-transformation plasmid was analyzed by Western Blot technique.1)Through the analysis of hormone degradation level of knockout mutants by HPLC,it was found that the testosterone degradation ability of wild type C.T.bacteria decreased significantly,and the degradation rate of wild type C.T.was 99%.The degradation rate of 3,17?-HSD was 6%,and the degradation rate of 3?-HSD was 30%;2)The polyclonal antibodies of 3,17?-HSD and 3?-HSD were successfully prepared,and their titers were 1:200 and 1:320000,respectively.The protein electrophoresis results of immunoprecipitation showed that the protein eluted with the two target proteins was concentrated between 26?45KDa,and there were about 6 to 8interacting proteins;3)Western Blot analysis of the immunoprecipitation results showed that both3,17?-HSD and 3?-HSD combined with SDRy protein.The immunoprecipitation strip analysis results of 3,17?-HSD antibody and C.T.bacterial whole protein showed that,In C.T.bacteria,3,17?-HSD and SDRy were both eluted,further verifying that they are protein interactors;4)The results of co-transformation showed that the expression of 3,17?-HSD was decreased by 0.77 times under the action of TetR and 0.75 times under the action of LuxR,and the regulatory effect of Lys R was not obvious.The expression of3?-HSD increased 3.58 times under the action of TetR;the expression of 3?-HSD increased 5.61 times under the action of Lys R;the effect of LuxR was not obvious.These results provide an experimental basis for the study of interactions between proteins involved in hormone degradation in C.T.bacteria.