Prokaryotic Expression And Comparation Of In Vitro Characters Of Human And Zebrafish PKA Alpha Catalytic Subunit Proteins

Protein kinase A（PKA）is a widely expressed serine（Ser）/threonine（Thr）kinase involved in various intracellular signaling events.Previous studies in our laboratory have shown that the fungicide tebuconazole can inhibit the biosynthesis of estrogen E₂ in zebrafish,thus causing reproductive endocrine disrupting effects on zebrafish.Early experiments based on H295R cells in vitro and the surface plasma resonance（SPR）test of the combination of TEB and hPKAcαshowed that TEB could inhibit the activity of PKA by binding withPKAcα,thus interfering with the intracellular c AMP/PKA signaling pathway,inhibiting the expression of aromatase CYP19A,and producing anti-estrogen effect.Although the primary structure of PKAcαis highly conserved in evolutionary terms（94%homology between hPKAcαand zPKAcα）,the binding reaction of tebuconazole to hPKAcαdoes not fully reflect its to zPKAcα.Therefore,in this study,hPKAcαand zPKAcαwere obtained by prokaryotic expression,and on this basis,their charactors in vitro were compared.The specific results are as follows:（1）Prokaryotic expression vectors p EASY-Blunt E2-PRKACA and p ET-28a-prkacaa were constructed to obtain active hPKAcαand zPKAcαproteins in vitro.After optimizing expression conditions,the soluble recombinant human hPKAcαand recombinant zPKAcαproteins were induced by 0.5 mmol/L IPTG,respectively under 25℃,4 h and 25℃,6 h induction conditions.PKA enzyme activity kit was used to confirm that both recombinant proteins had protein kinase activity,which could be used in following in vitro experiments.The enzyme activity of the two recombinant proteins was compared in vitro.It was found that the activity of the recombinant purified hPKAcαwas relatively higher,while the inhibition effect of the PKA specific inhibitor H89 was more obvious.（2）Anti–zPKAcαantibody was obtained by immunizing with a high titer（1:1024000）,which was verified by WB analysis to be effective in recognizing zPKAcα.The immune affinity of the commercial anti-hPKAcαpolyclonal antibody and the prepared anti-zPKAcαpolyclonal antibody against both hPKAcαand zPKAcαwas investigated by immunoassay respectively.The WB and ELISA results showed that both antibodies could recognize the two recombinant proteins.The logarithmic curve of OD₄₅₀-concentration of the two PKAcαantigens was linearly fitted,and the antigenic concentration of the two PKAcαantigens was compared when OD₄₅₀=1.0.It was found that C_zPKAcα:C_hPKAcα=1.075 in the anti-hPKAcαantigenic curve.In the curve of anti-zPKAcαantibody,C_hPKAcα:C_zPKAcα=1.950.The difference of immune affinity between the same antibody and the two recombinant PKAcαproteins in ELISA test was not significant,which proved that the two proteins had a certain crossover in the immune reaction.（3）With the help of homology modeling technology,the 3D structure model of zPKAcαwas established.The highest homology relatively few species of PKAcαstructures were selected as templates from PDB database,including the structure of hPKAcα.Discovery Studio software was used for 3D structure homology modeling of zPKAcαand assessment of the optimization.The most reasonable one was choosed for following test.The molecular docking technique was used to simulate the binding of tebuconazole to zPKAcαand hPKAcα,respectively.Compared with the positive control PKA inhibitor H89,the results showed that tebuconazole had certain binding ability with both of the PKAcαproteins.In conclusion,this study proved that hPKAcαand zPKAcαhave similar immune affinity in antigen-antibody reactions,and simulated results showed that both of the two kinds of proteins have certain binding properties with TEB,which laid a theoretical foundation for further exploration of the binding of TEB and PKAcαproteins.