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Heterologous Expression, Purification and Characterization of an Oligopeptidase A from the Pathogen Leptospira interrogans

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Abstract

Oligopeptidases are enzymes involved in the degradation of short peptides (generally less than 30 amino acids in size) which help pathogens evade the host defence mechanisms. Leptospira is a zoonotic pathogen and causes leptospirosis in mammals. Proteome analysis of Leptospira revealed the presence of oligopeptidase A (OpdA) among other membrane proteins. To study the role of oligopeptidase in leptospirosis, the OpdA of L. interrogans was cloned and expressed in Escherichia coli with a histidine tag (His-tag). The protein showed maximum expression at 37 °C with 0.5 mM of IPTG after 2 h of induction. Recombinant OpdA protein was purified to homogeneity using Ni-affinity chromatography. The purified OpdA showed more than 80% inhibition with a serine protease inhibitor but the activity was reduced to 30% with the cysteine protease inhibitor. The peptidase activity was increased significantly in the presence of Zn2+ at a neutral pH. Inhibitor assay indicate the presence of more than one active sites for peptidase activity as reported with the OpdA of E. coli and Salmonella. Over-expression of OpdA in E. coli BL21 (DE3) did not cause any negative effects on normal cell growth and viability. The role of OpdA as virulence factor in Leptospira and its potential as a therapeutic and diagnostic target in leptospirosis is yet to be identified.

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Acknowledgements

Authors acknowledge the financial support from Department of Science and Technology, Government of India (SR/S0/HS/078/2012) and Department of Higher education, Govt of Kerala, India.

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  1. Padikara K. Satheeshkumar

    Present address: Centre for Advanced Studies in Botany, Institute of Science, Banaras Hindu University, Varanasi, UP, India

Authors and Affiliations

  1. Department of Biotechnology, Interuniversity Centre for Genomics and Gene Technology, University of Kerala, Trivandrum, Kerala, India

    Prasannan V. Anu, Ananthakrishnan J. Nair, Gangaprasad A. Nair, Govinda Pillai M. Nair, Perumana R. Sudhakaran & Padikara K. Satheeshkumar

  2. Regional Medical Research Centre (ICMR) PB No: 13, Port Blair, Andaman and Nicobar Islands, India

    Madathiparambil G. Madanan

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Anu, P.V., Madanan, M.G., Nair, A.J. et al. Heterologous Expression, Purification and Characterization of an Oligopeptidase A from the Pathogen Leptospira interrogans. Mol Biotechnol 60, 302–309 (2018). https://doi.org/10.1007/s12033-018-0073-8

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