Mycobacterium 5'methylthioadenosine Nucleosidase, 5'MTAP Expression, Identification And Structural Analysis

Objective Cloning and expression of Rv0091and Rv0535encoding protein in Mycobacterium tuberculosis.Enzyme assay method was established to investigate the biochemical characters in detail, and identification and characterization of the enzyme active binding sites.Methods Construct the Rv0091and Rv0535prokaryotic expression plasmid,the vector was transformed into E. coli strain BL21trxB. After induced by IPTG, recombinant protein was purified by Ni2+-NTA chromatography and analyzed for purity by SDS-PAGE gels stained with Coomassie Blue. Immunological activity was identified by Western blot.The recombinant protein molecular weight was identified by Mass spectrometry. The enzyme-coupled assay detectes enzyme activity. Structure model was obtained by homology modeling.Results The expression plasmid was constructed and expressed in E. coli. BL21trxB, and the optimum expression system was conformed. The purity of the recombinant protein was more than95%. Western blot analysis confirmed that recombinant protein is one of Mycobacterium tuberculosis proteins. Mass spectrometry identified the relative molecular weight and theoretical molecular weight is basically the same. Enzyme assay showed the recombinant protein able to catalyze the substrate MTA. Enzymatic properties showed that the optimal buffer for the phosphate and hepes buffer, the poor thermal stability of the enzyme, the optimal temperature of37℃, optimal pH8-12, when the PH≤7, the protein denaturation and loss of some vitality. The homologous model and the sequence alignment results showed the substrate binding sites of MTAN and MTAP were identified.Conclusions we have cloned, expressed and purified the MTAN and MTAP, established the method for enzymatic assay and obtained the structure models of MTAN-FMA and MTAP-MTA binary complexes through homology modeling, combining with the sequence alignment, the enzymatic active sites have been deduced. These works pave the way for further structure-based design and screen of lead compounds.