Enzymatic Analysis Of M. Tuberculosis CysE

Tuberculosis (TB) caused by Mycobacterium tuberculosis has reemerged as a public health threat since the 80th of last century. Present anti-TB treating methods are not effective for TB since the synergy between HIV and tuberculosis, and the increasing of multi-drug resistant strains of M. tuberculosis. It has been over 40 years since a new drug for tuberculosis has been discovered, so it is emergent to provide new drug targets and develop effective anti-TB drugs.Serine acetyltransferase (CysE) catalyses the O-acetylation of L-serine using acetyl CoA as the acyl donor, which is the first step in a two-step enzymatic pathway for L-cysteine biosynthesis in bacteria and plants but not found in mammals. The first reaction products include CoASH and O-acetyl-L-serine which can be converted to L-cysteine by the catylation of O-acetylserine sulfhydrylase. Serine acetyltransferase (CysE) required for mycobacterial normal growth can be considered as a new drug target.The objectives of this study:(1) To amplify Tb cysE gene by PCR using the genomic DNA of M.tuberculosis H37Rv strain as template; (2) To clone PCR product of Tb cysE gene into a cloning vector pMD18-T for sequencing; (3) To subclone Tb cysE gene into an expression vector pET29b to construct pET29b-Tb cysE; and to express Tb CysE protein in E.coli BL21(DE3) under the induction of IPTG; (4) To purify CysE protein by affinity chromatography and identify CysE protein by SDS-PAGE and Western blotting; (5) To establish the method for detecting the activity of CysE enzyme; (6) To optimize the reaction conditions of CysE and determine the kinetic constants Km and Vmax of CysE. The results are followings:1. Tb cysE gene was amplified from M. tuberculosis H37Rv genomic DNA by polymerase chain reaction (PCR).DNA sequence of Tb cysE gene (690 bp) was acquired from M. tuberculosis genome database (http://genolist.pasteur.fr./TubercuList/). One set of primers was designed based on the sequence, including Ndel site and Xhol site which were added to 5'end of upstream primer and downstream primer respectively. Tb cysE gene was amplified from M. tuberculosis H37Rv genomic DNA by LA Taq DNA polymerase with high fidelity.2. pMD18-Tb cysE was constructed and Tb cysE was sequencedThe purified PCR product was ligated into pMD18-T plasmid, and NovaBlue competent cells were transformed with the ligation reaction. The positive recombinant plasmid pMD18-Tb cysE was confirmed by digestion of restriction endonucleases and the Tb cysE gene was sequenced. The results showed that there was no change of the cloned Tb cysE gene sequence compared with Tb cysE gene sequence.3. Expression vector pET29b-Tb cysE was constructed.pMD18-Tb cysE was digested by Ndel and Xhol, then cysE gene fragment was purified and ligated into the Ndel and Xhol site of vector pET29b to generate pET29b-Tb cysE. The C-terminus of CysE protein was fused with histidine tag in pET29b vector.4. CysE protein was expressed in E. coli BL21 (DE3) and purified by histidine-Ni2+affinity chromatography.pET29b-Tb cysE were transformed into E. coli BL21 (DE3) competent cells. BL21 (DE3) cells carrying pET29b-Tb cysE were induced with IPTG. The induced cells were broken by sonication, and the proteins from both supernatant and pellet fractions were analyzed by SDS-PAGE. The results showed that Tb CysE protein in E. coli BL21 (DE3) was soluble expressed.Tb CysE protein was purified by Ni+affinity chromatography. The elution fraction 1 (1ml) was quantified as 0.037mg/ml by coomassie brilliant blue method. The purified CysE protein was identified by SDS-PAGE and Western blotting.5. The enzyme assay of CysE was established.The amount of product CoASH can be obtained by adding DTNB (Ellman's Reagent) into the reaction system and then recording the OD value at 405nm showed by microplate reader.6. Kinetic parameters of CysE protein were determined.(1) Initial velocity was determined.The reaction was performed with L-serine, AcCoA and different concentrations of CysE at 37℃for different time. The curve of velocity versus concentration of CysE enzyme and the curve of concentration of product CoASH versus time were plotted. The results showed that the concentration of CysE was 0.74ug/ml and the incubation time was 5 minutes within the initial velocity.(2) The optimal reaction conditions of CysE were determined.The reaction was performed at different temperature and pH in the intial velocity. The optimal temperature of CysE is 37℃, and the optimal pH is 7.5, and Mg2+is not essential for the catalytic activity of CysE.(3) The kinetic parameters of CysE were determined.Under the optimal reaction conditions, the reaction was performed with one substrate saturated while the other one at several different concentrations. Km and Vmax were caculated by the double-reciorocal plot method. The Km and Vmax for AcCoA is 0.051335+0.00499mM and 0.008189±0.00047mM-1min, respectively, for L-Serine, the Km is 0.026405±0.00063mM and Vmax is 0.006455±0.00047 mM-1min.Conclusions:In this study, we constructed pET29b-Tb cysE expression vector and expressed soluble recombinant CysE protein in E.coli BL21(DE3) and purified CysE protein. We established the enzyme assay of CysE, and obtained the kinetic constants of CysE under the optimal reaction conditions.