Overexpression Of M.tuberculosis Phosphoglucosamine Mutase In E.coli BL21(DE3)

Tuberculosis (TB) caused by infection of Mycobacterium tuberculosis has become Global infectious diseases. It severely threats the public health and kills more people in the world than any other single bacterial infection. It is called white plague. Major problem is the emergence of multi-drug and extra-drug resistant strains makes invalid of many anti-TB drugs. Thus, more efficient anti-TB drugs are desperately needed. The mycobacterial cell wall is required for the survival and growth of mycobacteria in the host, and has the unique components and structures. The mybacterial cell wall core consists of mycolic acids, arabinogalacan (AG), and peptidoglyc- an. AG is attached to the peptidoglycan via a linker disaccharide,α-L-rha- mnosyl- (1→3) -α-D-N-acetyl-glucosaminosyl-1-posphate. The function of phosphoglucosamine mutase is to catalyze glucosaminosyl-6-posphate into glucosaminosyl-1-posphate in biosynthesis path way of UDP-N- acetyl -glucosamine, a donor of N-acetyl-glucosamine for the formation of the disaccharide linker. The reaction of glucosaminosyl-6-posphate to glu- cosaminosyl-1-posphate does not exist in human, therefore, phosphogluco- samine mutase is an ideal target for developing new anti-TB drugs. In order to determine the crystal structure of phosphoglucosamine mutase and target design the enzymatic inhibitor, it is essential to obtain enough GlmM protein. Therefore, we design this project and carry out an experiment. First the Tb glmM gene encoding phosphoglucosamine mutase needs to be cloned and then expressed to soluble GlmM protein in E.coli. We obtain enough GlmM protein by separation and purification.The objectives of this study are:To clone glmM gene from M.tuberculosis H37Rv genome and construct pEHisTEV-Tb glmM expression vector; to overexpress GlmM protein in E.coli BL21(DE3) and purify GlmM protein for further analyzing crystal structure of phosphoglucosamine mutase and studying the kinetics of phosphoglucosamine mutase.The methods used in this study are:(1) To amplify Tb glmM gene encoding phosphoglucosamine mutase by polymerase chain reaction (PCR) from the genomic DNA of M.tubercu- losis H37Rv strain; (2) To clone PCR product of Tb glmM gene into a cloning vector pSTBlue 1 for sequencing; (3) To subclone Tb glmM gene to an expression vector pEHisTEV to construct pEHisTEV-Tb glmM and overexpress Tb GlmM protein in E.coli BL21(DE3); (4) To test expressed GlmM protein by SDS-PAGE.Followings are results we got in this study:1. Tb glmM gene was amplified from M. tuberculosis H37Rv genomic DNA by polymerase chain reaction (PCR).DNA sequence of Tb glmM gene (1347 bp) was acquired from M. tuberculosis genome database (http://genolist.pasteur.fr/TubercuList/). One set of primers was designed based on the sequence, and NcoI site and HindIII site were add to 5'end of upstream primer and downstream primer respectively. Tb glmM gene was amplified from M. tuberculosis H37Rv genomic DNA by Expand HF Enzyme Mix.2. pSTBlue 1-Tb glmM was constructed and Tb glmM was sequenced.The end conversion of PCR product was ligated into pSTBlue 1 plasmid, and DH5αcompetent cells were transformed with the ligation reaction. Recombiant plasmid pSTBlue 1-Tb glmM was confirmed by digestion of restriction endonucleases. Tb glmM was sequenced and sequencing data was analyzed by Multalin research against M. tuberculosis genome database. The result showed that there was no any change of base in cloned Tb glmM gene.3. pEHisTEV-Tb glmM expression plasmid was constructedpSTBlue 1-Tb glmM was digested by NcoI and HindIII, Tb glmM gene was purified and ligated into the NcoI and HindIII sites of plasmid pEHisTEV resulting in pEHisTEV-Tb glmM. pEHisTEV-Tb glmM was confirmed by digestion of restriction endonuclease, The N-terminus of GlmM protein was fused with His-tag (6 Hisitidines) and TEV (tobacco etch virus) protease recognization sequence in pEHisTEV-Tb glmM. His-tag is used for purification GlmM protein and the TEV protease recognization sequence is used for removing His-tag (6 Hisitidines).4. Tb GlmM protein was expressed in E.coli BL21(DE3).BL21(DE3) competent cells were transformed with pEHisTEV-Tb glmM, and BL21(DE3) competent cells were transformed with pEHisTEV as a control. Tb GlmM protein was induced under IPTG. Induced BL21(DE3) cells were French Pressed and both supernatant and pellet fractions were analyzed by SDS-PAGE. The result showed that Tb GlmM protein was overexpressed in E.coli BL21(DE3).Followings are conclusions we got in this study:1. Tb glmM gene was successfully amplified from M. tuberculosis H37Rv genomic DNA by Expand HF Enzyme Mix and pEHisTEV-Tb glmM expression plasmid was constructed.2. GlmM protein was overexpressed in E.coli BL21(DE3). The soluble GlmM protein was purified by Ni2+ affinity chromatograpy.The purified GlmM protein will be utilized to determine the crystal structure of phosphoglucosamine mutase and study its kinetics.