Expression And Identification Of M.tuberculosis WbbL Protein In E.coli BL21(DE3)

Tuberculosis (TB) caused by the infection of Mycobacteriumtuberculosis is the first killer for human beings. Major problem is theimpressive emergence and wide distribution of multidrug-resistant strainsof M. tuberculosis. Since present anti-TB drugs are not effective for TB,the mortality resulting from infection of M. tuberculosis has beenincreasing globally in recent years. Therefore, it is obvious that moreeffective anti-TB drugs are needed. The targets of new drugs need to be validated during drug discovery.The mycobacterial cell wall is required for the survival and growth ofmycobacteria in the host, and has the unique components and structures.The mycolated arabinogalactan are covalently attached to peptidoglycanvia the disaccharide linker (L-rhamnosyl-N-acetyl-glucosaminyl-phosphate)to build up the core structure of mycobacterial cell wall. Therefore, thedisaccharide linker is fundamental to the structural integrity of the cell wall.The function of rhamnosyl transferase is to add the rhamnosyl residue ofdTDP-L-rhamnose into N-acetyl-glucosaminyl-phosphate to form thedisaccharide linker. The essentiality of rhamnosyl transferase has beenproved, therefore, rhamnosyl transferase is a potential drug target todevelop more effective anti-TB drugs. Our long-term objective is to establish a molecule model of screeninganti-TB drugs. Therefore, the Tb wbbL gene encoding rhamnosyltransferase needs to be cloned and expressed to soluble WbbL protein inE.coli. Soluble WbbL protein will be utilized for further purification andstudy of kinetics of rhamnosyl transferase as well as development ofenzyme assay to screen anti-Tb drugs. The whole genome of Mycobacterium tuberculosis H37Rv strainhas been sequenced. H37Rv genome database provides a basis forcloning any target gene. Tb wbbL gene is Rv3265 in H37Rv genome. The objectives of this study are: (1) To amplify Tb wbbL gene encoding rhamnosyl transferase bypolymerase chain reaction (PCR) from the genomic DNA ofM.tuberculosis H37RV strain; (2) To clone PCR product of Tb wbbLgene into a cloning vector pMD18-T for sequencing; (3) To subcloneTb wbbL gene to an expression vector pET16b to construct pET16b-Tb wbbL; and to overexpress Tb WbbL protein in E.coli BL21(DE3)under different induction conditions; (4) To establish theco-expression system for expressing chaperons of pKJE7 plasmidand soluble Tb WbbL protein in E.coli BL21(DE3) under differentinduction conditions; (5) To test expressed WbbL protein by SDS-PAGEand Western blot methods. Followings are results we got in this study: 1. Tb wbbL gene was amplified from M. tuberculosis H37Rvgenomic DNA by polymerase chain reaction (PCR). DNA sequence of Tb wbbL gene (906 bp) was acquired from M.tuberculosis genome database (http://genolist.pasteur.fr/TubercuList/).One set of primers was designed based on the sequence, and Ndeâ… site andBamHâ… site were added to 5' end of upstream primer and downstreamprimer respectively. Tb wbbL gene was amplified from H37Rv genomicDNA by LA Taq DNA polymerase with high-fidelity. 2. pMD18-Tb wbbL was constructed and Tb wbbL was sequenced. The PCR product was ligated into pMD18-T plasmid, and NovaBluecompetent cells were transformed with the ligation reaction. Recombiantplasmid pMD18-Tb wbbL was confirmed by digestion of restrictionendonucleases and PCR. Tb wbbL was sequenced and sequencing data wasanalyzed by BLAST research anainst M. tuberculosis H37Rv genomedatabase. The result showed that there was no any change of base incloned Tb wbbL gene. 3. pET16b-Tb wbbL expression plasmid was constructed. pMD18-Tb wbbL was digested by Ndeâ… and BamHâ… , Tb wbbLfragment was purified and ligated into the Ndeâ… and BamHâ… sites ofplasmid pET16b resulting in pET16b-Tb wbbL. pET16b-Tb wbbL wasconfirmed by digestion of restriction endonuclease and PCR. TheN-terminus of WbbL protein was fused with His-tag (10 Hisitidines) inpET16b-Tb wbbL. His-tag is used for detection of WbbL protein byWestern blot and purification of WbbL protein by Ni2+ affinitychromatography. 4. Tb WbbL protein was expressed...