Abstract：Objective To clone and express the Mycobacterium tuberculosis trptophan synthase αsubunit ( MtTrpA) gene trpAand to study the role of MtTrpA. Methods The trpA gene was amplified by PCR from Mycobacterium tuberculosis H37Rv straingenomic DNA and cloned into a prokaryotic expression vector pET30a. The resulting recombinant expression plasmid pET30a - trpAwas then transformed into the E. coli strain DH5αand a high-level expression E. coli BL21 ( DE3) was established after induction with IPTG. SDS - PAGE and MALDI - TOF determined the relative molecular weight of this recombinant protein His - rMtTrpA. Itssecondary and 3D structures were determined by circular dichroism and homologous modeling. The enzyme studies tested the functionsof MtTrpA. Results The Mycobacterium tuberculosis trpA gene ( 813 bp) and highly purified recombinant His - rMtTrpA protein wereobtained. The relative molecular weight of recombinant His - rMtTrpA protein was determined to be 33. 151 ×103 ( vector included) . Secondary structure of His - rMtTrpA had about 31. 8 % α helix, 31. 8% β sheet, 8. 4 % β turn, 27. 9% random coil at 25℃.Homologous modeling shows His - rMtTrpA as ( β/ α) 8 - barrel protein. His - rMtTrpA can most effectively activate βreaction whenmolecular ratio of MtTrpA and MtTrpB was 2. 2. Conclusion This study obtained purified His - rMtTrpA; Functional analysis pavedthe way for further design of inhibitors against this enzyme.
