Cloning, Expression And Characterization Of Rv3813c Gene From Mycobacterium Tuberculosis

The complete genome sequence and annotation of Mycobacteriumtuberculosis H37Rv was published in 1998, and the genome sequence wasreannotated in 2002. H37Rv genome database provides a basis forunderstanding the pathogenic mechanism and developing new targets ofanti-tuberculosis drugs. H37Rv genome contains 4411532 base pairsincluding 4044 genes, and these genes are divided into 11 functionalclassifications. Two hundred and seventy-two genes encode proteins ofunknown function and 1051 genes encode conserved hypotheticalproteins. The cell wall of M. tuberculosis is a structural barrier for them tosurvive and multiplicate in the host, and the cell wall is a good target fordeveloping anti-tuberculosis drugs. Therefore, identification of moreproteins and enzymes involved in cell wall metabolism will be helpful forus to understand the details of the cell wall metabolism and to find moregood targets for anti-tuberculosis drug discovery. The arabinogalactan (AG) is unique structure of Mycobacterial cellwall, and AG contains gaclatan that consists of D-Galf and arabinan thatconsists of D-Araf. The donor of D-Araf is β-D-Arabinofuranosyl-1-Phosphoryl-Decaprenol (DPA), however, the details of DPA formation arenot clear so far. Three enzymes, transferase, phosphatase and epimerase,may involve in the proposed pathway of DPA. Huang, et al. identified M.tuberculosis Rv3806c gene encoding transferase. Ma cloned 2 candidatesfor the gene encoding M. tuberculosis epimerase and is investigating theepimerase activity of candidates. Therefore, it is necessary for us to identifyphosphatase for further understanding biosynthetic pathway of DPA. Rv3813c encodes a conserved hypothetical protein. Rv3813c proteinwas analyzed by using NCBI Conserved Domain Search Tool andConserved Domain Architecture Retrieval Tool. The results show thatRv3813c protein contains conserved domains which are present inhydrolase and phosphate. The objectives of this study were (1) To amplify the PCR product ofRv3813c gene from M tuberculosis genomic DNA; (2) To clone Rv3813cgene to pSTBlue1 resulting in pSTBlue1-Rv3813c and to sequenceRv3813c; (3) To construct an expression vector pET29b-Tb Rv3813c; (4)To optimize the express of Rv3813c protein in E.coli BL21(DE3); (5) Topurify Rv3813c protein and identify the function of Rv3813c protein. Followings are results we got in this study: 1. Rv3813c gene from H37Rv genomic DNA was amplified. DNA sequence of Rv3813c was acquired from M. tuberculosis H37Rvgenome database (http://genolist.pasteur.fr/Tuberculist/). One set primerswas designed for Rv3813c gene. The NdeI site was added at 5' end ofupstream primer, Rv3813c-1, and XhoI site was added at 5' end ofdownstream primer, Rv3813c-2. Rv3813c gene was amplified fromH37Rv genomic DNA by Vent DNA polymerase. 2. Rv3813c gene was cloned to generate pSTBlue1-Tb Rv3813c Rv3813c gene was ligated to the site of pSTBlue1 to generatepSTBlue1-Tb Rv3813c.pSTblue-Rv3813c (5) was sequenced andsequencing data was analyzed by comparing to Rv3813c gene from H37Rvgenome database. The result showed that there was no any base change incloned in Rv3813c gene. 3. Expression plasmid pET29b-Rv3813c was constructed. pSTBlue1-Rv3813c (5) was digested by NdeI and XhoI.Rv3813c genewas purified and ligated to the NdeI and XhoI sites of an expressionplasmid pET29b to construct pET29b-Rv3813c.The N-terminus ofRv3813c gene was fused with (histidine)6 tag in pET29b. The fusionprotein can be detected by Western blot and purified by affinitychromatography. 4. Rv3813c protein was expressed in BL21(DE3). pET29b-Rv3813c (5) was transformed to BL21(DE3) andpET29b-Rv3813c(5)/BL21(DE3) was induced by 1 mM IPTG at 37°C.Expressed Rv3813c protein was detected by SDS-PAGE and identified byWestern blot. 5. Rv3813c protein was purified and phosphatase activity of Rv3813cprotein was detected. (1) Rv3813c protein was purified by using HIS-Select HF NickelAffinity Gel, and the purified Rv3813c protein was confirmed by Westernblot. NaCl and imidazole in purified Rv3813c protein were removed b...