Expression And Functional Analysis Of M. Tuberculosis WecA Membrane Protein

Mycobacterium tuberculosis is the pathogen of tuberculosis, which infected nearly one third of worldwide populations. The incidence is rising yearly due to the emergence of multi-drug and even extra-drug resistant strains. Therefore, identifying new drug targets of anti-tuberculosis may become a major task currently.M. tuberculosis has a unique cell wall structure which plays an important role on its existence and reproduction. The core structure of mycobacterial cell wall consists of mycolic acids, arabinogalacan and peptidoglycan. Arabinogalacan is attached to peptidoglycan via a linker disaccharide L-rhamnose-D-N-acetylglucosamine. Therefore,the linker disaccharide is essential for the integrality of cell wall. The biosynthesis of the linker disaccharide consists of two steps catalyzed by two enzymes. In the first enzymatic reaction, N-acetylglucosamine-1-phosphate (GlcNAc-1-P) group of UDP-GlcNAc is added to C50-P (Decaprenyl phosphate) to form C50-P-P-GlcNAc, which is catalyzed by N-acetylglucosamine-1-phosphate transferase. Obviously, this transferase plays the key role on the synthesis of the linker disaccharide.In E. coli, N-acetylglucosamine-1-phosphate transferase (WecA) encoded by wecA gene involves in synthesis of lipopolysaccharide (LPS). In our preliminary studies, Rv1302 has been identified to be wecA gene coding M. tuberculosis WecA, which complemented on LPS synthesis of E. coli MV501, a wecA gene defective strain. M. semgmatis wecA gene knockout strain was constructed. The growth curves, cell morphological and structural changes of M. semgmatis wecA gene knockout strain clarified the essentiality of wecA gene for mycobacterial growth. Therefore, WecA is a good potential target to develop anti-tuberculosis drugs.To study catalytic mechanism of M. tuberculosis WecA protein it is necessary to acquire enough M. tuberculosis WecA protein. M. tuberculosis WecA protein was predicted as membrane protein with 11 transmembrane domains. Due to its complicated structure, it is difficult to over-express the WecA membrane protein in E. coli strains and purify.The objectives of this study:1. To express WecA membrane protein in E. coli ER2566 strain by pET16b-Tb wecA expression vector. 2. To establish methods of detecting enzyme activity of WecA protein, including TLC and HPLC. 3. To attempt other methods of WecA protein expression. To construct pCold-Tb wecA expression plasmid and express WecA protein in E. coli MV501 strain at low temperature. To construct pET29b-Tb wecA del1 and pET29b-Tb wecA del2 expression vectors and to express N-truncated WecA proteins by using three E. coli strains. To express WecA protein by using MembraneMax Protein Expression Kit in vitro.The results are followings:1. WecA membrane protein was expressed by pET16b-Tb wecA expression vector in E. coli ER2566 strain and purified.WecA protein was induced by IPTG (1 mM) at 25℃for 16 hours in E. coli ER2566 cells carrying plasmid pET16b-Tb wecA. The membrane fraction was prepared and WecA protein was solublized by detergent DDM (n-Dodecyl-β-D-maltoside). WecA protein was analyzed by Dot blot, SDS-PAGE and Western blot. The results showed that WecA protein was detected in the membrane protein fraction of ER2566 carrying plasmid pET16b-Tb wecA. The N-terminus of WecA protein was fused with His-tag in pET16b vector, therefore, WecA protein was purified by histidine-Ni2+ affinity chromatography. Enzyme assay was performed by using purified WecA protein.2. The methods to detect enzyme activity of WecA protein, including TLC and HPLC, were established.(1) The enzymatic activity of WecA was detected by TLC.Substrates C50-P and UDP-GlcNAc were incubated with purified WecA protein. The reaction was extracted by chloroform/methanol/ammonia mixture. The organic phase was vacuum dried. The samples were dissolved in chloroform and loaded on TLC plate. TLC was used to detect the consumption of C50-P and the production of C50-P-P-GlcNAc. The purified WecA protein showed activity of N-acetylglucosamine-1-phosphate transferase. (2) The enzymatic activity of WecA was detected by HPLC. Substrates C50-P and UDP-GlcNAc were incubated with purified WecA protein. The reaction was extracted by chloroform/methanol/ammonia mixture. HPLC was used to detect the consumption of UDP-GlcNAc in the aqueous phase. The peak area of UDP-GlcNAc was reduced obviously after incubation of substrates C50-P and UDP-GlcNAc with purified WecA protein. It indicated that WecA protein had N-acetylglucosamine-1-phosphate transferase activity.3. Other methods of WecA expression were attempted.(1) WecA membrane protein was expressed by pCold-Tb wecA expression vector in E. coli MV501 strain at low temperature.pET16b-Tb wecA was digested by NdeⅠa nd BamHⅠa nd the Tb wecA gene was ligated into the NdeⅠand BamHⅠsites of pColdⅡvector, a cold-shock expression vector, to generate pCold-Tb wecA. The promoter of cold-shock gene cspA can enhance the stability and solubility of target protein. The N-terminus of WecA protein was fused with His-Tag in pColdⅡvector.WecA protein was induced by 1 mM IPTG at 15℃for 24 hours in E. coli MV501 strain carrying plasmid pCold-Tb wecA. The supernatant, pellet and membrane protein fractions were analyzed by SDS-PAGE and Western blot. WecA protein was detected in the membrane protein fraction of MV501 carrying plasmid pCold-Tb wecA.(2) Expression of N-truncated WecA proteins in E. coli The deleted Tb wecA gene at the 5'end, Tb wecA del1 and Tb wecA del2, were acquired through PCR and pMD18-Tb wecA del1 and pMD18-Tb wecA del2 cloning vectors were constructed. The Tb wecA del1 and Tb wecA del12 were confirmed by DNA sequencing.pMD18-Tb wecA del1 and pMD18-Tb wecA del2 were digested by NdeⅠand XhoⅠ. Tb wecA del1 and Tb wecA del2 were ligated into the NdeⅠand XhoⅠsites of vector pET29b to generate pET29b-Tb wecA del1 and pET29b-Tb wecA del2 expression vectors respectively. The C-terminus of truncated WecA proteins was fused with His-tag in pET29b vector.pET29b-Tb wecA del1 and pET29b-Tb wecA del2 were transformed into three different E. coli stains BL21(DE3), ER2566 and CD41(DE3) respectively. Expression of the truncated WecA proteins was induced by 1 mM IPTG. The supernatant, pellet and membrane protein fractions were analyzed by SDS-PAGE and Western blot. However, the truncated WecA proteins were not detected. It indicated that the amino acids at N-terminus may be required by WecA protein.(3) In vitro expression of WecA membrane proteinpET16b-Tb wecA, pET29b-Tb wecA del1 and pET29b-Tb wecA del2 plasmids as templates were added into in vitro expression system, MembraneMax Protein Expression Kit, respectively. After initiation reaction for 30 minutes and feed reaction for 2 hours, the protein products were analyzed by SDS-PAGE and Western blot. Neither full-length WecA membrane protein nor truncated ones were detected. In vitro expression of WecA protein needs to be optimized in the further study.Conclusions:1. WecA membrane protein was expressed by pET16b-Tb wecA vector in E. coli ER2566 strain and purified by affinity chromatography.2. N-acetylglucosamine-1-phosphate transferase assay by TLC and HPLC was established. N-acetylglucosamine-1-phosphate transferase activity of purified WecA protein was detected. Therefore, it is confirmed that Rv1302 is wecA gene coding Mycobacterium tuberculosis N-acetylglucosamine-1-phosphate transferase (WecA).3. Other expression methods of WecA membrane protein were tried. WecA membrane protein was expressed by pCold-Tb wecA in E. coli MV501, however, the amount of WecA membrane protein was still low. pET29b-Tb wecA del1 and pET29b-Tb wecA del2 expression vectors were constructed, but the truncated WecA proteins were not expressed. It indicates that N-terminal amino acids are important to maintain the structure of WecA protein. In vitro expression of WecA membrane protein by using MembraneMax Protein Expression Kit was tried.