Identification Of M.tuberculosis Rv1018c

Tuberculosis (TB) drug resistance and the spread of Mycobacterium tuberculosis in AIDS patients in several outbreaks of TB epidemic led to a rebound in the global situation, since present anti-TB drugs are not effective for TB, the mortality resulting from infection of M. tuberculosis has been increasing globally in recent years. So, there is an urgent need to develop effective anti-TB drug.The mycobacterial cell wall is required for the survival and growth of mycobacteria in the host, the core of the cell wall consists of peptidoglycan, arabinogalactan, and mycolic acid for connection. The mycolated arabinogalactan are covalently attached to peptidoglycan via the disaccharide linker (L-rhamnosyl-N-acetyl-glucosaminyl-phosphate) to build up the core structure of mycobacterial cell. Thus, the common moity of the peptidoglycan and disaccharide joint molecule is N-acetyl glucosamine, so the factors affecting its synthesis can be as ideal targets of new drugs.M. tuberculosis Rv1018c has 37% homology with E. coli GlmU, which is a bifunctional enzyme for the UDP-GlcNAc synthesis. The glucosamine- 1-phosphate acetyl transferase activity catalyzes the formation of N-acetylglucosamine-1-phosphate (GlcNAc-1-P) from glucosa mine-1- phosphate (GlcN-1-P) using acetyl Co-A as the acetyl donor, and N-acetylglucosamine-1-phosphate uridyltransferase activity catalyzes the formation of UDP-N-acetyl glucosamine (UDP-GlcNAc) from GlcNAc-1-P and UTP. UDP-GlcNAc is a precusor for the synthesis of disaccharide linker and peptidoglycan.In order to identify Tb Rv1018c, we need to clone Tb Rv1018c (glmU) gene and to overexpress Tb GlmU protein in E.coli BL21(DE3) for further detecting Tb GlmU activities by high performance liquid chromatogram (HPLC).The objectives of this study: (1) To amplify Tb glmU gene by PCR from the genomic DNA of M.tuberculosis H37RV strain; (2) To clone PCR product of Tb glmU gene into a cloning vector pBluescript II KS(-) for sequencing; (3) To subclone Tb glmU gene to an expression vector pET16b to construct pET16b-Tb glmU; and to overexpress Tb GlmU protein in E.coli BL21(DE3) under different induction conditions; (4) To purify GlmU protein by affinity chromatography and identify GlmU protein by SDS-PAGE and Western blotting; (5) To identify the function of GlmU protein by HPLC.Followings are results we have got in this study:1. Tb glmU gene was amplified from M. tuberculosis H37Rv genomic DNA by PCR.DNA sequence of Tb glmU gene (1488 bp) was acquired from M.tuberculosis genome database (http://genolist.pasteur.fr/TubercuList/). One set of primers was designed based on the sequence, and NdeⅠsite and Xho I site were added to 5'end of upstream primer and downstream primer respectively. Tb glmU gene was amplified from M.tuberculosis H37Rv genomic DNA by Vent DNA polymerase with high-fidelity.2. pBS-Tb glmU was constructed and Tb glmU was sequenced.The purified PCR product was ligated into pBluescript II KS(-) plasmid, and DH5αcompetent cells were transformed with the ligation reaction. The positive recombinant plasmid pBS-Tb glmU was confirmed by digestion of restriction endonucleases and the Tb glmU gene was sequenced.The results showed that the cloned glmU gene does not have any wrong bases.3. pET16b-Tb glmU expression plasmid was constructed pBS-Tb glmU was digested by Nde I and Xho I, then glmU gene fragment was purified and ligated into the Nde I and Xho I sites of vector pET16b to generate pET16b-Tb glmU. The N-terminus of GlmU protein was fused with histidine tag in pET16b plasmid.4. Tb GlmU protein was expressed in E.coli BL21(DE3) and purifid. pET16b-Tb glmU were transformed into BL21(DE3) competent cells. BL21(DE3) cells carring pET16b-Tb glmU were induced with IPTG . The induced cells were broken by sonication, and the proteins from both supernatant and pellet fractions were analyzed by SDS-PAGE. The results showed that Tb GlmU protein in BL21(DE3) was soluble expressed.Tb GlmU protein was purified by Ni2+affinity chromatography. The elution fraction 2 (1 ml) was quantified as 0.533 mg/ml by coomassie brilliant blue method. The purified GlmU protein was identified by SDS-PAGE and Western blotting.5. Tb GlmU protein activity was determined by HPLCThe reaction was performed in 50 mM Tris (pH8.2) containing glucosamine-1-phosphate (GlcN-1-P), acetyl CoA and purified Tb GlmU at 37℃for 30 minutes. The result showed there was an absorption peak of N-acetylglucosamine-1-phosphate (GlcNAc-1-P). It proved that the purified GlmU protein has the glucosamine-1-phosphate acetyl transferase activity.The reaction was performed in 50 mM Tris (pH8.2) containing UDP-N-acetyl glucosamine (UDP-GlcNAc), pyrophosphoric acid and purified Tb GlmU at 37℃for 30 minutes. The result showed there was an absorption peak of N-acetylglucosamine-1-phosphate (GlcNAc-1-P). It was proved that the purified GlmU protein has the N-acetylg lucosa- mine-1-phosphate uridyltransferase activity.Conclusions:In this study, we constructed pET16b-Tb GlmU expression vector and overexpressed considerable soluble recombinant Tb GlmU protein in E.coli BL21(DE3). The purified GlmU protein was assayed by HPLC. M. tuberculosi GlmU protein shows bifunctional enzyme activities, including the glucosamine-1-phosphate acetyl transferase activity and N-acetylg lucosamine-1-phosphate uridyltransferase activity. The purified GlmU protein will be useful to establish the method to screen the inhibitors to GlmU enzyme.