Expression And Enzymatic Property Analysis Of M. Tuberculosis MurB

Mycobacterium tuberculosis is often called bacillus tuberculosis, which causes tuberculosis. The presence and prevalence of drug-resistant strain (especially the multi-resistant bacterium) as well as HIV/AIDS and the large floating population in the late 1980s caused the outbreak of tuberculosis worldwide. The incidence of tuberculosis were on the rise in many countries. Tuberculosis is still a serious infectious disease endangering human's health by now and because of which a new generation of anti-tuberculosis drug research is needed.Mycobacterial cell wall is the structural foundation for the survival, so the cell wall of Mycobacterium tuberculosis can be developed as a new drug target. The core of the cell wall consists of peptidoglycan, arabinogalactan, and mycolic acid. If the cell wall of Mycobacterium tuberculosis would be destroyed, the germ will die. Because peptidoglycan is not found in humans, peptidoglycan is a potential drug target to develop more effective anti-TB drugs. Mycobacterium tuberculosis murA-G genes encoding the enzyme MurA-G participated in the eight steps synthesis of peptidoglycan from the substrate UDP-N-acetylglucosamine (UNAG). So anyone of the seven enzymes can be the target of developing the new anti-tuberculosis drug.Uridine diphospho-N-acetylenolpyruvylglucosamine reductase (MurB) is needed during the second step of the synthesis of peptidoglycan, to the in-depth study MurB activity, we must first clone the gene murB which encodes the enzyme MurB and then express soluble MurB protein in E.coli. Soluble MurB will be utilized for further purification and study the kinetics of MurB as well as development of enzyme assay to screen anti-Tb drugs. The objectives of this study:(1) To amplify Tb murB gene by Polymerase Chain Reaction (PCR) from the genomic DNA of M. tuberculosis H37Rv strain; (2) To clone PCR product of Tb murB gene into a cloning vector pMD18-T for sequencing; (3) To subclone Tb murB gene to an expression vector pET29b to construct pET29b-Tb murB; and to overexpress Tb MurB protein in E.coli BL21(DE3)/pKJE7 under different induction conditions; (4) To purify MurB protein by affinity chromatography and identify MurB protein by SDS-PAGE and Western blotting; (5) To establish a method to detect the activity of MurB enzyme; (6) To optimize the reaction conditions of MurB and determine the kinetics constants of the MurB enzyme.Followings are results we have got in this study:1. Tb murB gene was amplified from M. tuberculosis H37Rv genomic DNA by polymerase chain reaction (PCR).DNA sequence of Tb murB gene (1110 bp) was acquired from M.tuberculosis genome database (http://genolist.pasteur.fr./TubercuList/). One set of primers was designed based on the sequence, and Nde I site and Xho I site were added to 5'end of upstream primer and downstream primer respectively in order to clone Tb murB gene into the Nde I site and Xho I site in a cloning vector pET29b. Tb murB gene was amplified from H37Rv genomic DNA by LA Taq polymerase with high-fidelity.2. pMD18-Tb murB was constructed and Tb murB was sequenced.The purified PCR product was ligated into pMD18-T plasmid, and NovaBlue competent cells were transformed with the ligation reaction. Recombiant plasmid pMD18-Tb murB was confirmed by digestion of restriction endonucleases BamH I. Tb murB was sequenced and sequencing data was analyzed by BLAST research anainst M. tuberculosis H37Rv genome, the results were conformed and showed that Tb murB amplified by the PCR method was the correct gene and could encode the Tb MurB protein theoretically.3. Expression vector pET29b-Tb murB was constructed.pMD18-Tb murB was digested by Nde I and Xho I, then murB gene fragment was purified and ligated into the Nde I and Xho I sites of vector pET29b to generate pET29b-Tb murB. Recombiant plasmid pET29b-Tb murB was confirmed by digestion of restriction endonucleases Apa I. The C-terminus of MurB protein was fused with histidine tag in pET29b vector.4. MurB protein was expressed in E.coli BL21(DE3)/pKJE7 and purified by histidine-Ni2+affinity chromatography.pET29b-Tb murB were transformed into BL21(DE3)/pKJE7 competent cells. BL21(DE3)/pKJE7 cells carring pET29b-Tb murB were cultured for 4h,25℃to make the cells get into the logarithmic growth phase. Then they were induced with L-arabinos and the final concentration of the L-arabinose was 0.5mg/ml. Then the cells had been cultured for 3h,25℃in order to express molecular chaperones dnaK,dnaJ and grpE. Then cells were induced with IPTG with the final concentrition 0.4 mM, cells had been cultured for 8h,25℃to induce BL21(DE3)/pKJE7 to express recombination protein. BL21(DE3)/pKJE7 germ after induction were crashed by ultrasonic. Total proteins from both supernatant and pellet fractions were analyzed by SDS-PAGE. The results showed that soluble MurB protein was produced in BL21(DE3)/pKJE7 cells.MurB protein was purified by histidine-Ni2+affinity chromatography. The elution fraction 1 (1 ml) was quantified as 64.42μg/ml by Coomassie brilliant blue method. The purified MurB protein was identified by SDS-PAGE and Western blotting.5. The enzyme assay of MurB was established.The reaction was performed in 50 mM Tris-HCl (pH8.0) containing UDP-N-acetylenolpyruvylglucosamin, NADPH, coenzyme FAD and purified MurB at 37℃for 30 minutes, the reaction was stopped by being put into ice water mixture. Then 754 spectrophotometer was used to measure the optical density at 340 nm, the significantly reducesed optical density at 340 nm indicated that there was product producted and the purified MurB protein has enzyme activity.6. The optimal reaction conditions of MurB enzyme was determined.The reaction was performed with different temperature and different pH in the initial velocity (the concentration of Tb MurB was 1μg/ml and the incubation time was 10 minutes). The product was monitored by 754 spectrophotometer at 340nm. The result showed that the optimal temperature of MurB enzyme is 37℃and the optimal pH is 8.0. 7. The kinetic parameters of MurB enzyme were determined.The enzyme reactions were performed with different concentration of substrates in the optimal reaction conditions of MurB (37℃; pH8.0; 1μg/ml of MurB;5 minutes). The product was monitored by 754 spectrophotometer at 340 nm. Km and Vmax of MurB were calculated by the double-reciprocal plot method. For substrate Uridinediphospho-N-acetylenolpyruvylglucosamine, Km=0.3752 mmol/L, Vmax=0.6876 mmol/(L.min).Conclusions:In this study, we constructed pET29b-Tb murB expression vector and overexpressed considerable soluble recombinant MurB protein in E.coli BL21(DE3)/pKJE7. The method to test enzyme activity of MurB was developed. The reaction condition of MurB was optimized and Km and Vmax were determined. MurB enzyme assay will be utilized in screening inhibitors of MurB enzyme to develop the anti-TB drugs in the future.