Cloning, expression and enzymatic analysis of the recombinant tryptophan synthase αsubunit from Mycobacterium tuberculosis H37Rv

ZHAO Jing-jing; XU Sheng-feng; WANG Hong-jun; SHEN Hong-bo; WANG Hong-hai

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Journal of Microbes and Infections ›› 2008, Vol. 4 ›› Issue (4) : 195-199.

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Cloning, expression and enzymatic analysis of the recombinant tryptophan synthase αsubunit from Mycobacterium tuberculosis H37Rv

ZHAO Jing-jing; XU Sheng-feng; WANG Hong-jun; SHEN Hong-bo; WANG Hong-hai

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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.

Key words

Mycobacterium tuberculosis / Tryptophan synthase / αsubunit / Homologous modeling

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ZHAO Jing-jing; XU Sheng-feng; WANG Hong-jun; SHEN Hong-bo; WANG Hong-hai. Cloning, expression and enzymatic analysis of the recombinant tryptophan synthase αsubunit from Mycobacterium tuberculosis H37Rv[J]. Journal of Microbes and Infections. 2008, 4(4): 195-199