Optimal Expression And Function Analysis Of M. Tuberculosis MurA

Mycobacterium tuberculosis is the pathogen that causes tuberculosis. As a global issue, tuberculosis (TB) is still an infectious disease with extremely high morbidity and mortality, catched by nearly one thirds of worldwide populations, moreover the incidence is rising yearly. Major problem is the emergence of multi-drug and extra-drug resistant strains makes invalid of many anti-TB drugs. Therefore, identifying new targets from M. tuberculosis itself and developing new anti-tuberculosis drugs may become a chief task currently.The core of the mycobacterial cell wall consists of mycolic acid, arabinogalactan, and peptidoglycan for connection. One of the best known and most validated targets for antibacterial therapy is the machinery for peptidoglycan biosynthesis. The biosynthetic pathway of Mycobacterium tuberculosis peptidoglycan is a complex two-stage process. The first stage, which occurs in the cytoplasm, is the formation of the monomeric building block N-acetylglucosamine-N-acetylmuramyl pentapeptide. The first committed step in the pathway is the transfer of an enolpyruvate residue from phosphoenolpyruvate (PEP) to position 3 of UDP-N-acetylglu-cosamine. This reaction is catalysed by the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA). MurA is ubiquitous to both Gram-positive and Gram-negative bacteria, and has no homologue in mammalian cells. Therefore, MurA is a potential target to develop anti-TB drugs.Our long-term objective is to establish a molecule model of screening anti-TB drug. Therefore, we must first clone the gene murA which encodes the enzyme MurA, and then express soluble MurA protein. Soluble MurA will be utilized for further purification and study the kinetics of MurA as well as development of enzyme assay to screen anti-Tb drugs.The objectives of this study:(1) to express M. tuberculosis MurA protein by pET29b express vector in E.coli BL21(DE3) and optimize the expression conditions in order to obtain the soluble MurA; (2) to separate and solubolize inclusion body-forming MurA protein and purify MurA protein by affinity chromatography. To identify the purified MurA protein by SDS-PAGE and Western blotting and prepare anti-MurA polyclonal antibody by injecting rabbit with purified MurA protein and detect the serum specificity and its titer; (3) to express M. tuberculosis MurA protein by pVV16 express vector in M.smegmatis mc2155; (4) to establish methods to detect the activity of MurA enzyme.The results are followings:1. Expression vector pET29b-Tb murA was constructed.pMD18-Tb murA was digested by Ndeâ… and Hindâ…¢, then Tb murA gene fragment was purified and ligated into the Ndeâ… and Hindâ…¢sites of vector pET29b to generate pET29b-Tb murA. The C-terminus of MurA protein was fused with histidine tag in pET29b vector.2. Tb MurA protein was expressed in E.coli BL21(DE3) and the expression conditions was optimized.pET29b-Tb murA were transformed into BL21(DE3) competent cells. BL21(DE3) cells carring pET29b-Tb murA were induced. Induced BL21(DE3) cells were sonicated and both supernatant and pellet fractions were analyzed by SDS-PAGE and Western blotting. MurA protein was expressed in E.coli BL21(DE3),but they were insoluble in inclusion body. Optimize the expression conditions, for example, to reduse the concen-tration of IPTG, to induce under lower temperature condition and to coexpress with chaperons, and so on. The Tb MurA still formed inclusion bodies.3. Inclusion body-forming Tb MurA protein was separated, solubolized, purified and detected.The inclusion body-forming Tb MurA protein was separated, solubolized with different buffers and obtained lots of soluble Tb MurA protein. Tb MurA protein was purified by histidine-Ni2+ affinity chromatography. The elution fraction was quantified by coomassie brilliant blue method. The purified MurA protein was confirmed by SDS-PAGE and Western blotting.4. Anti-Tb MurA polyclonal antibody was prepared and identified.Purified Tb MurA was prepared with particular means for injecting rabbit. Antiserum was separated and assayed for antibody titer as 1:78125 by enzyme-linked immunosorbent assay. Tb MurA protein expressed in mc2155 and protein of M. smegmatis mc2155 was detected by antiserum followed by antirabbit-IgG-conjugated with alkaline phosphatase. The result showed anti-MurA polyclonal antibody had high specificity.5. Expression vector pVV16-Tb murA was constructed.pMD18-Tb murA was digested by Ndeâ… and Hindâ…¢, then murA gene fragment was purified and ligated into the Ndeâ… and Hindâ…¢sites of vector pVV16 to generate pVV16-Tb murA. The C-terminus of Tb MurA protein was fused with histidine tag in pVV16 vector.6. Tb MurA protein in M. smegmatis mc2155 was expressed and identified.pVV16-Tb murA was transformed into M. smegmatis mc2155 competent cells to express the Tb MurA protein. The mc2155 cells were broken by sonication, and the proteins from both supernatant and pellet fractions were analyzed by SDS-PAGE and Western blotting. The results showed that Tb MurA protein in mc2155 was soluble expressed.7. Tb MurA protein was purified by affinity chromatography.The C-terminus of Tb MurA was fused with histidine tag in pVV16, therefore Tb MurA was purified by histidine-Ni2+ affinity chromatography. The elution fraction was quantified by coomassie brilliant blue method. The purified Tb MurA protein was confirmed by SDS-PAGE and Western blotting.8. The enzyme assay of Tb MurA was established.(1) HPLC:UDP-G1cNAc was separated with Nova-Pak C18 at a flow rate of 0.5mLmin-1 under 20 mM triethylamine-acetic acid buffer (pH 4.0) and was monitored at 260 nm. The peak of UDP-G1cNAc was appeared at 6.3 min approximately. Its reduction indicated that MurA has activity. (2) Chemical colorimetric assays:The inorganic phosphate produced in the reation was detected. The reaction of inorganic phosphate and ammonium molybdate results in the formation of molybdenum blue, which turns malachite green from yellow to blue. The OD value at 630 nm was detected.Conclusions:In this study, we constructed pET29b-Tb murA expression vector and overexpressed recombinant Tb MurA protein in inclusion body and anti-MurA polyclonal antibody was prepared.We constructed pVV16-Tb murA expression vector and expressed considerable soluble recombinant Tb MurA protein in M. smegmatis mc2155.We established Tb MurA enzyme assay and identified Tb MurA enzyme activity.