Cloning, Expression And Characterization Of Gene Rv3807c From Mycobacterial Tuberculosis

Tuberculosis (TB) is one of communicabe diseases, which threaten the public health. Mycobacterium tuberculosis is the pathogen for tuberculosis. The total length of Mycobacterium tuberculosis H37Rv strain genome is 4411532bp, including 4404 genes. The functions of the proteins from 272 genes are unknown, and 1051 genes encode for are conserved hypothetical proteins.The cell wall of mycobacterium is the important barrier for the survival of Mycobacterium tuberculosis, and the cell wall is the target for developement of new drugs against TB. Mycobacterial cell wall consists of inner layer peptidoglycan (PG), middle layer arabinogalactan (AG) and outer layer mycolic acid. AG is an important structural polysaccharades. The active donor of D-arabinose is decaprenyl-phospho- arabinose (DPA). Currently, protein functions of Rv3806c, Rv3790 and Rv3791 have been studied and confirmed. The specific synthetic pathway of DPA is elucidated gradually. The pathway includes three sequential reactions. First, 5-phosphoribosyl 1-pyrophosphate (PRPP) is transferred by the Rv3806 5-phosphoribosyltransferase to decaprenyl phosphate, forming 5-phosphoribosyl- monophospho-decaprenol (5-P-DPR). Second, 5-P-DPR is dephosphorylated by phospholipid phosphatase, forming decaprenyl-phospho-ribose (DPR). Last, DPR by the heteromeric Rv3790/Rv3791 epimerase leads to the formation of the decaprenyl- phosphor-arabinose (DPA). In this pathway, the phosphatase has not being identified in the second step. Rv3807c, sharing the same promoter with Rv3806c, encodes for a protein with 166 amino acid and it is an unknown function membrane protein. Bioinformatics study of protein structural domain indicates Rv3807c as phospholipids phosphatase. It is postulated that 5-P-DPR is dephosphorylated to DPR by Rv3807c. The objectives of the study are (1) clone Rv3807c from Mycobacterium tuberculosis; (2) Overexpression protein of Rv3807c in E.coli Er2566(DE3); (3) Characterize the function of the protein encoded by Rv3807c.The methods and results of the experiment in this study are:1. Rv3807c was amplified from Mycobacterium tuberculosis genomic DNA by polymerase chain reaction (PCR).2. Cloning plasmid of pMD18T-Rv3807c was transformed into Novablue competent cells, and digest the plasmid by restriction enzymes.3. Correct DNA sequencing of pMD18T-Rv3807c plasmid with BLAST.4. Expression plasmid pET16b-Rv3807c and pCold-Rv3807c was constructed by restriction enzymes NdeI and BamHI.5. Expression plasmid pET16b-Rv3807c was transformed into E.coli BL21(DE3) and Er2566(DE3) competent cells, and expression plasmid pCold-Rv3807c was transformed into E.coli BL21(DE3) competent cells. They were detected by SDS-PAGE and Western Blotting.6. Soluble Rv3807c proteins in E.coli were purified by Ni-NTA affinity chromato- graphy.7. Membrane protein encoded by Rv3807c was abstracted with ultracentrifuge, and was detected by SDS-PAGE and Western Blotting.8. Activity of Rv3807c proteins assay TLC analysis the changing of the products 5-P-DPR and DPR after incubation of Rv3807c protein with DP and PRPP as well as the E. coli membrane containing expressed Rv3806cIn this study, we cloned Rv3807c from Mycobacterium tuberculosis and detected the function of expressed Rv3807c in E.coli.