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Expression, Purification and Characterisation of Secreted Esterase Rv2525c from Mycobacterium tuberculosis

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Abstract

Rv2525c from Mycobacterium tuberculosis belongs to the domain of unknown function (DUF) 1906 superfamily, but it also contains the motif G-X-S-X-G, the consensus active site sequence of the ester/lipid family. Biochemical analysis indicated that the mature Rv2525c protein is secreted. The discovery and characterisation of novel enzymes secreted by M. tuberculosis are vital for understanding the pathogenesis of the most important human bacterial pathogen. The proteome of M. tuberculosis contains over 400 potentially secreted proteins, of which the majority remain uncharacterised. In this study, we cloned and expressed the rv2525c gene in Escherichia coli and purified the recombinant protein using a three-step process (affinity chromatography, ion exchange chromatography, gel filtration chromatography), obtaining more than 99 % pure protein. Mass spectrometry was performed to confirm that the purified protein was Rv2525c. Circular dichroism spectroscopy results showed that its conformation was stable at pH ranging from 6.0 to 8.0 and at temperatures ≤40 °C. Moreover, we tested the esterase activity using p-nitrophenyl esters (C2, C4, C6, C8, C12, C14, C16). This enzyme exhibited broad substrate acceptance, preferentially hydrolysing p-nitrophenyl butyrate (C4) at pH 7.0 and 37 °C. The dynamic activity test demonstrated that the optimal conditions were pH 8.0 and 38 °C. Site-directed mutagenesis studies revealed that Gly 113, Ser 115 and Gly 117 residues play catalytic roles in Rv2525c.

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Acknowledgments

This work was supported by the Major State Basic Research Development Program of China (973 program, no. 2012CB518800), the National Natural Science Foundation of China (nos. 31201920 and 31272538), and the State Key Laboratory of Veterinary Biotechnology Research Fund (no. SKLVBP201417).

Conflict of Interest

The authors have declared that no competing interests exist.

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    Authors and Affiliations

    1. Division of Bacterial Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 427 Maduan Street, Nangang Dist., Harbin, 15000, People’s Republic of China

      Guanghui Dang, Liping Chen, Zhaoli Li, Yingying Cui, Jun Cao, Shenye Yu & Siguo Liu

    2. School of Medicine, Tsinghua University, Beijing, 100084, China

      Xiaoxia Deng & Hai Pang

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    1. Guanghui Dang
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    2. Liping Chen
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    Correspondence to Hai Pang or Siguo Liu.

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    Guanghui Dang and Liping Chen contributed equally to this work.

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    Dang, G., Chen, L., Li, Z. et al. Expression, Purification and Characterisation of Secreted Esterase Rv2525c from Mycobacterium tuberculosis . Appl Biochem Biotechnol 176, 1–12 (2015). https://doi.org/10.1007/s12010-015-1555-9

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