Cloning, Overexpression And Characterization Of M.smegmatis MSMEG₅243 And MSMEG₆086 Genes

Mycobacterium tuberculosis, a species of the genus Mycobacteria is the pathogen of tuberculosis. The mycobacterial cell wall is a unique structure for mycobacterial survival and growth in the host. The core of mycobacterial cell wall consists of inner layer peptidoglycan (PG), middle layer arabinogalactan (AG) and outer layer mycolic acid. AG is an important structure to connect PG and mycolic acid and to keep the integrity of the cell wall. Besides,D-arabinan of AG doesn't exist in mammal,but in mycobacterium. Therefore, AG is an ideal target to develop new effective anti-TB drugs.Mycobacterium smegmatics(M.smegmatis) is also a species of the genus Mycobacteria. Nonpathogenic Mycobacterium smegmatics and Mycobacterium tuberculosis have similar structure of cell wall, therefore, they may have similar enzymes involved in biosynthesis of cell wall. Dr.Xin found an endo-arabinase from Mycobacterium smegmatics in 1996, which degraded AG. Endo-arabinase is studied to develop new anti-TB drugs because it can destroy the cell wall of Mycobacterium smegmatics. The arabinase was purified and analyzed with mass spectrum (MS) by Xu Dong in DaLian Medical University. After analyzed with bioinformatics, DNA sequences of candidate proteins were determined and analyzed preliminary. On the basis of the previous study, two candidate genes, MSMEG₅243 and MSMEG₆086, will be cloned and overexpressed to identify the relation to endo-arabinase.The objectives of this study are (1) To clone MSMEG₅243 and MSMEG₆086 from M.smegmatis; (2) To over-express proteins of MSMEG₅243 and MSMEG₆086 in both E.coli BL21(DE3) and M.smegmatis; (3) To characterize the function of proteins encoded by MSMEG₅243 and MSMEG₆086.Followings are the experimental methods and results we got in thisstudy:1. MSMEG₅243 and MSMEG₆086 were amplified from M.smegmatis mc2155 genomic DNA by polymerase chain reaction (PCR). Cloning plasmid of pMD18-MSMEG₅243 and pMD18-MSMEG₆086 were constructed.2. Expression plasmid pVV2-MSMEG₅243 and pVV2-MSMEG₆086 were electroporated into M.smegmatis mc2155 competent cells. Expression plasmid pET16b-MSMEG₅243 and pET16b-MSMEG₆086 were transformed into E.coli BL21(DE3) competent cells.3. The expression of MSMEG₅243 and MSMEG₆086 in M.smegmatis mc2 155 and E.coli BL21(DE3) were detected by SDS-PAGE and Western Blotting.4. Soluble MSMEG₅243 and MSMEG₆086 proteins in E.coli BL21(DE3) were purified by Ni-NTA affinity chromatography and purified MSMEG₅243 and MSMEG₆086 proteins have been detected by SDS-PAGE and Western Blotting.5. Activity of MSMEG₅243 and MSMEG₆086 proteins assay(1) The variation of arabinose in M.smegmatis mc2155 where MSMEG₅243 and MSMEG₆086 were overexpressed was detected with HPLC. The results showed the ratio of arabinose in overexpressed strains was similar to that in wild type.(2) The morphological characteristics of overexpressed M.smegmatis mc2 155 was studied by SEM. The form of wild M.smegmatis mc2155 is usually rod. Compared with wild M.smegmatis mc2155, The morphology of strain with with overxpression of MSMEG₅243 has been changed individually. While the strain with with overxpression of MSMEG₆086 was keep its morphology.(3)The effect of the overexpression of MSMEG₅243 and MSMEG₆086 on the cell growth of M.smegmatis mc2155 was analyzed by adsorption spectrophotometry method. The growth curve showed the rate of overexpressed M.smegmatis mc2155 is slower than wild type.(4)Enzyme assay:AG substrate was incubated with MSMEG₅243 and MSMEG₆086 proteins expressed in E.coli BL21(DE3) and products were separated by bio-gelA1.5M column. Eluent was detected by The results showed arabinase activity of MSMEG₅243 and MSMEG₆086 proteins were not detected。Conclusions:In this study, we cloned MSMEG₅243 and MSMEG₆086 from M.smegmatis and detected expressed MSMEG₅243 and MSMEG₆086 in M.smegmatis mc2155 and in E.coli BL21(DE3).At the same time, we studied the relation between overexpressed prroteins and arabinase. In conclusions, arabinase activity of overexpressed prroteins was not found. However, the results will be utilized in further study on arabinase and corresponding genes.