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Eniyan K, Bajpai U, Powell A, et al. Cloning, expression, purification and bioinformatic analysis of 2-methylcitrate synthase from Mycobacterium tuberculosis. Asian Pac J Trop Med. 2015;8(1):19-23doi: 10.1016/S1995-7645(14)60181-4.
Eniyan, K., Bajpai, U., Powell, A., & Dainty, A. (2015). Cloning, expression, purification and bioinformatic analysis of 2-methylcitrate synthase from Mycobacterium tuberculosis. Asian Pacific journal of tropical medicine, 8(1), 19-23. https://doi.org/10.1016/S1995-7645(14)60181-4
Eniyan, Kandasamy, et al. "Cloning, expression, purification and bioinformatic analysis of 2-methylcitrate synthase from Mycobacterium tuberculosis." Asian Pacific journal of tropical medicine vol. 8,1 (2015): 19-23. doi: https://doi.org/10.1016/S1995-7645(14)60181-4
Eniyan K, Bajpai U, Powell A, Dainty A. Cloning, expression, purification and bioinformatic analysis of 2-methylcitrate synthase from Mycobacterium tuberculosis. Asian Pac J Trop Med. 2015 Jan;8(1):19-23. doi: 10.1016/S1995-7645(14)60181-4. PMID: 25901919.
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Asian Pac J Trop Med. 2015 Jan;8(1):19-23. doi: 10.1016/S1995-7645(14)60181-4.

Cloning, expression, purification and bioinformatic analysis of 2-methylcitrate synthase from Mycobacterium tuberculosis.

Asian Pacific journal of tropical medicine

Kandasamy Eniyan, Urmi Bajpai, Powell, Dainty

Affiliations

  1. Department of Biomedical Science, Acharya Narendra Dev College (University of Delhi), Govind Puri, Kalkaji, New Delhi 110019, India.
  2. Department of Biomedical Science, Acharya Narendra Dev College (University of Delhi), Govind Puri, Kalkaji, New Delhi 110019, India. Electronic address: [email protected].

PMID: 25901919 DOI: 10.1016/S1995-7645(14)60181-4

Abstract

OBJECTIVE: To clone, express and purify 2-methylcitrate synthase (Rv1131) gene of Mycobacterium tuberculosis (M. tuberculosis) and to study its structural characteristics using various bioinformatics tools.

METHODS: Rv1131 gene was amplified by polymerase chain reaction using M. tuberculosis H37Rv genomic DNA and cloned into pGEM-T easy vector and sequenced. The gene was sub-cloned in pET28c vector, expressed in Escherichia coli BL21 (E. coli BL21) (DE3) cells and the recombinant protein was identified by Western blotting. The protein was purified using Nickel affinity chromatography and the structural characteristics like sub-cellular localization, presence of transmembrane helices and secondary structure of the protein were predicted by bioinformatics tools. Tertiary structure of the protein and phylogenetic analysis was also established by in silico analysis.

RESULTS: The expression of the recombinant protein (Rv1131) was confirmed by western blotting using anti-HIS antibodies and the protein was purified from the soluble fraction. In silico analysis showed that the protein contains no signal peptide and transmembrane helices. Active site prediction showed that the protein has histidine and aspartic acid residues at 242, 281 & 332 positions respectively. Phylogenetic analysis showed 100% homology with major mycobacterial species. Secondary structure predicts 2-methylcitrate synthase contain 51.9% alpha-helix, 8.7% extended strand and 39.4% random coils. Tertiary structure of the protein was also established.

CONCLUSIONS: The enzyme 2-methylcitrate synthase from M. tuberculosis H37Rv has been successfully expressed and purified. The purified protein will further be utilized to develop assay methods for screening new inhibitors.

Copyright © 2015 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

Keywords: 2–methylcitrate synthase; Expression; Mycobacterium tuberculosis; Propionyl–CoA; Purification

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