Study On Cloning And Expression Properties Of Malate Synthetase Glcb Gene

Tuberculosis is a chronic communicable disease caused by Mycobacterium tuberculosis and spread around the world.It becomes persistent in them and sustains life through the glyoxylate bypass.As malate synthetase(MS), encoded by Glcb gene,is a key ratelimting enzyme in the glyoxylate bypass and a decisive factor in mycobacterial persistce, therefore this study was to the key enzyme as the research object。In order to obtain recombinant protein with enzymatic activities of malate synthetase,The Glcb gene was amplified by polymerase chain reaction(PCR) from Myobacterium tuberculosis H37 Rv strain genomic DNA and cloned into pET28-a(+) vector, named pET28-glcb. The recombinant protein was expressed in E.coli BL21(DE3). In order to improve the expression quantity of the recombinant malate synthetase in this experiment,three factors for the optimization of induced culture were studied, including induced time,inducer concentration and inducing temperature. The experimental result shows that, the expression quantity of malate synthetase is up, when the induction time was 8 h, inducer concentration of induction was 0.6 mM and induction temperature was 20 ℃. The results show that we can get the higher quantity of malate synthetase by optimization of the induction conditions. The expressed Glcb was further purified on a Ni-NTA resin affinity chromatography. The target protein was eluted with 200 mmol/L imidazole buffers, by SDS-PAGE, won the high purity of MS recombinant proteins. The value of GlcB enzyme activity was determined by realtime monitoringthe change of light absorption value at 418 nm.The final computation results was enzyme activity of 6.6 mmol/(mg·min).The recombined MS was used as the target, peptides and MS protein crystal(3S9I)were done molecular simulate docking by the Discovery Studio 2.1, the final docking results were determined the following two 7-peptides, respectively named and amino acid sequence are: No.3: RMHSSYH; No.113: PSREPPN. Finally, the tow 7-peptides were synthesized by the solid-phase synthesis of Fmoc,and detected by the mass spectrum. The detection results are correct. Two 7-peptides were respectively reactioned with MS, and had inhibitory effect on MS. The test showed that peptide 113 had strongest effects with the inhibitory rate of 68.01%, peptide 3 with 63.98%.This study provided lead compounds for the research of new peptide anti-TB drugsa,and the development of the novel drugs against Mycobacterium tuberculosis.