Cloning,Expression,and Characterization Of MSMEG-6402Gene From Mycobacterium Smegmatis

Mycobacterium tuberculosis is the pathogen for tuberculosis. Currently, due to thecontinuous increasing of multidrug-resistant tuberculosis (MDR-TB), and the lack ofclinical anti-TB drugs, it turns hard to cure tuberculosis again. Therefore, identifyingnew targets from M. tuberculosis genome and developing new anti-tuberculosis drugsbecome to be a chief task currently.The cell wall core of Mycobacterium tuberculosis is termed themycolyl-arabinagalactan-peptidoglycan (mAGP) complex, which is essential for theviability of M. tuberculosis. The arabinogalactan is the major cell wall polysaccharide ofmycobacteria. This unique nature leads to the conclusion that the enzymes thatsynthesize this structure should yield a number of potential drug targets. Furthermorethe immediate precursor of the polymerized Araf is decaprenylphosphoryl-D-arabinose(DPA). DPA is synthesized initially from the transfer of phosphribosyl pyrophosphate(PRPP) to decaprenyl phosphate (DP) to form5-phospho-decaprenylphospho-ribose(5-p-DPR) via the5-phosphorobosyl transferase (Rv3806c). Subsequently, the5-phosphate unit is removed from5-phospho-decaprenylphospho-ribose to formdecapreny-lphosphoryl-ribose (DPR) by the putative Rv3807phospholipid phosphatase.The last step of the biosynthetic pathway of decaprenyl-phospho-arabinose is the2’-epimerization from DPR to DPA, catalyzed by the heteromeric Rv3970/Rv39712’-epimerase. The function and characteristics of the5-phospho-α-D-ribose-1-pyrophosphate: decapronyl phosphate5-phosphoribosyltransferase (Rv3806c) and the downstream enzymedecaprenyl-phospho-ribose-2’-epimerase (Rv3790/Rv3791) are identified to be essentialfor synthesize of decapresyl-phospho-arabinose (DPA). However, the biologicalfunction of the Rv3807gene product, as a good candidate as a specificdecaprenyl-phospho-ribose-5’-phosphate phosphatase, which is located next to the phosphoribosyltransferase (Rv3806c), has not been accurately elucidated yet. In thisstudy, MSMEG-6402gene, as the homologous gene of Rv3807c gene, was cloned,expressed and purified. The functional study of MSMEG-6402protein will help toimprove the understanding of the DPA synthesis metabolic pathway and find newanti-TB drug targets.This research include:1) Expression plasmid pET16b-MSMEG-6402wasconstructed in E.coli BL21(DE3) for soluble expression, and MSMEG-6402proteinwas purified by histidine-Ni2+affinity chromatography. Enzyme assay was performedby using purified MSMEG-6402protein.2) As the lack of substrate5-P-DPR, Rv3806cprotein is used to complete enzyme activity detection. Rv3806c protein was expressedand purified by the same way used in the expression and purification of the MSMEG–6402protein. Both MSMEG-6402protein and Rv3806c protein were detected bySDS-PAGE and Western-blotting;3) According to the nature of the substrate DP/PRPPand the product DPPR/DPR, malachite green-ammonium molybdate assay, thin layerchromatography (TLC) and mass spectrometry (MS) were used to detected the enzymeactivity of the purified MSMEG–6402protein respectively.The results are as following:1) The Rv3806c and the MSMEG–6402genes were successfully expressed in E.coli BL21(DE3) and purified by Ni-NTA with the right molecular weight (33kDa,20kDa);2) The methods for detection of enzyme activity of MSMEG-6402protein wereestablished, including malachite green-ammonium molybdate, TLC and MS.The reaction of inorganic phosphate and ammonium molybdate results in theformation of molybdenum blue, which turns malachite green from yellow to blue. TheOD value at630nm was detected.Substrates DP and PRPP were incubated with purified MSMEG-6402protein fordetection. The samples were dissolved in chloroform and loaded on TLC plate. Theconsumption of DP/PRPP and the production of DPPR/DPR were detected after medicalcolorimetric assays. The purified MSMEG-6402protein showed activity ofphosphatase.MS was used to detect the consumption of DP/PRPP and the production ofDPPR/DPR. The peak area of DP/PRPP was reduced significantly after incubation withpurified MSMEG-6402protein and Rv3806c protein. Meanwhile, DPPR/DPR appeared corresponding peak areas. It indicated that MSMEG-6402protein had the phosphataseactivity.In this study, different methods were used to detect the phosphatase activity ofMSMEG-6402protein, catalyzing5-P-DPR to form DPR. In the future work, wewill improve the purification methods of MSMEG-6402protein, to reduce the contentof miscellaneous protein. The synthesis route of DPA in vitro will be constructed tocomplete the functional detection of the MSMEG-6402protein.