Expression, Purification And Kinetics Of M.tuberculosis RmlB

Tuberculosis (TB) caused by the infection of Mycobacterium tuberculosis is a serious infectious disease endangering human health. The main reason is that multi-drug resistant strains of mycobacterium tuberculosis increase, and the existing anti-TB drugs are not effective for tuberculosis, so each year there are 900 million new cases and 3 million people were killed, a new generation of anti-tuberculosis drug research is needed.Mycobacterial cell wall is the structural foundation for the survival, 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. The mycolic acid and arabinogalactan are covalently attached to peptidoglycan via the disaccharide linker (L-rhamnosyl-N- acety-l-glucosaminyl-phosphate). If the disaccharide linker was destroyed, the cell wall of Mycobacterium tuberculosis would be destroyed and the germ would die. Because L-rhamnose (L-Rha) is non-toxin to human and it is not found in humans, L-Rha is a potential drug target to develop more effective anti-TB drugs. Mycobacterium tuberculosis rmlA-D genes encoding the enzyme RmlA-D participated in the synthesis of dTDP-L-Rha from the substrate glucose-1-phosphate. So anyone of the four enzymes can be as the target of developing the new anti-tuberculosis drug.dTDP-D-glucose 4, 6-dehydrogenase (RmlB) is needed during the second step of the synthesis of dTDP-L-rhamnose, to the in-depth study RmlB activity, we must first clone the gene rmlB which encodes the enzyme RmlB, and then express soluble RmlB protein in E.coli. Soluble RmlB will be utilized for further purification and study the kinetics of RmlB as well as development of enzyme assay to screen anti-Tb drugs.The objectives of this study: (1) To amplify Tb rmlB gene by chain reaction (PCR) from the genomic DNA of M.tuberculosis H37RV strain; (2) To clone PCR product of Tb rmlB gene into a cloning vector pMD18-T for sequencing; (3) To subclone Tb rmlB gene to an expression vector pET29b to construct pET29b-Tb rmlB; and to overexpress Tb RmlB protein in E.coli BL21(DE3) under different induction conditions; (4) To purify RmlB protein by affinity chromatography and identify RmlB protein by SDS-PAGE and Western blotting; (5) To establish a method to detect the activity of RmlB enzyme; (6) To optimize the reaction conditions of RmlB and determine the kinetics constants of the RmlB enzyme. Followings are results we have got in this study:1. Tb rmlB gene was amplified from M. tuberculosis H37Rv genomic DNA by polymerase chain reaction (PCR).DNA sequence of Tb rmlB gene (996 bp) was acquired from M. tuberculosis genome database (http://genolist.pasteur.fr./TubercuList/). One set of primers was designed based on the sequence, and NdeI site and Xho I site were added to 5'end of upstream primer and downstream primer respectively. Tb rmlB gene was amplified from H37Rv genomic DNA by primer STARTMHS polymerase with high-fidelity.2. pMD18-Tb rmlB was constructed and Tb rmlB 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 rmlB was confirmed by digestion of restriction endonucleases. Tb rmlB was sequenced and sequencing data was analyzed by BLAST research anainst M. tuberculosis H37Rv genome database. The result showed that there was only one change of base in cloned Tb rmlB gene, but there was no effection in expression the right RmlB protein.3. expression vector pET29b-Tb rmlB was constructed.pMD18-Tb rmlB was digested by Nde I and Xho I, then rmlB gene fragment was purified and ligated into the Nde I and Xho I sites of vector pET29b to generate pET29b-Tb rmlB. The C-terminus of RmlB protein was fused with histidine tag in pET29b vector.4. RmlB protein was expressed in E.coli BL21(DE3) and purified by histidine-Ni2+ affinity chromatography.pET29b-Tb rmlB were transformed into BL21(DE3) competent cells. BL21(DE3) cells carring pET29b-Tb rmlB were induced with IPTG and total proteins from both supernatant and pellet fractions were analyzed by SDS-PAGE. The results showed that soluble RmlB protein was produced in BL21(DE3) cells. RmlB protein was purified by histidine-Ni2+ affinity chromatography. The elution fraction 1 (1 ml) was quantified as 1.553 mg/ml by coomassie brilliant blue method. The purified RmlB protein was identified by SDS-PAGE and Western blotting.5. The enzyme assay of RmlB was established.The reaction was performed in 50 mM HEPES (pH7.5) containing dTDP-Glucose, NAD+ and purified RmlB at 30℃for 30 minutes and the reaction was stopped by 1 M NaOH. Then 754 spectrophotometer was used to measure the optical density at 340 nm, the significantly increased optical density at 340 nm indicated that there was product producted. The purified RmlB protein has activity.6. The optimal reaction conditions of RmlB enzyme was determined.The reaction was performed with different temperature and different pH in the initial velocity (the concentration of Tb RmlB was 0.05mg/ml and the incubation time was 30 minutes). The product was monitored by 754 spectrophotometer at 340nm.The result showed that the optimal temperature of RmlB enzyme is 30℃and the optimal pH is 7.5.7. The kinetic parameters of RmlB enzyme were determined.The enzyme reactions were performed with different concentration of substrates in the optimal reaction conditions of RmlB (30℃; pH7.5; 0.05mg/ml of RmlB; 30 minutes). The product was monitored by 754 spectrophotometer at 340 nm. Km and Vmax of RmlB were calculated by the duble-reciprocal plot method. For substrate dTDP-D-glucose, Km = 3.965 mmol/L, Vmax = 4.2513 mmol/(L.min).Conclusions:In this study, we constructed pET29b-Tb rmlB expression vector and overexpressed considerable soluble recombinant RmlB protein in E.coli BL21(DE3). The method to test enzyme activity of RmlB was developed. The reaction condition of RmlB was optimized and Km and Vmax were determined. RmlB enzyme assay will be utilized in screening inhibitors of RmlB enzyme to develop the anti-TB drugs in the future.