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Cloning of Intron-Removed Enolase Gene and Expression, Purification, Kinetic Characterization of the Enzyme from Theileria annulata

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Abstract

Tropical theileriosis is a disease caused by infection with an apicomplexan parasite, Theileria annulata, and giving rise to huge economic losses. In recent years, parasite resistance has been reported against the most effective antitheilerial drug used for the treatment of this disease. This emphasizes the need for alternative methods of treatment. Enolase is a key glycolytic enzyme and can be selected as a macromolecular target of therapy of tropical theileriosis. In this study, an intron sequence present in T. annulata enolase gene was removed by PCR-directed mutagenesis, and the gene was first cloned into pGEM-T Easy vector and then subcloned into pLATE31 vector, and expressed in Escherichia coli cells. The enzyme was purified by affinity chromatography using Ni–NTA agarose column. Steady-state kinetic parameters of the enzyme were determined using GraFit 3.0. High quantities (~65 mg/l of culture) of pure recombinant T. annulata enolase have been obtained in a higly purified form (>95 %). Homodimer form of purified protein was determined from the molecular weights obtained from a single band on SDS-PAGE (48 kDa) and from size exclusion chromatography (93 kDa). Enzyme kinetic measurements using 2-PGA as substrate gave a specific activity of ~40 U/mg, K m: 106 μM, kcat: 37 s−1, and k cat/K m: 3.5 × 105 M−1 s−1. These values have been determined for the first time from this parasite enzyme, and availability of large quantities of enolase enzyme will facilitate further kinetic and structural characterization toward design of new antitheilerial drugs.

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Acknowledgments

This project was supported by Yildiz Technical University Scientific Research Projects Coordination Department under the Project Number: 2011-0704-KAP03.

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Authors and Affiliations

  1. Department of Bioengineering, Faculty of Engineering and Natural Sciences, Gaziosmanpasa University, 60240, Tokat, Turkey

    Ebru Cayir

  2. Department of Bioengineering, Faculty of Chemical and Metallurgical Engineering, Yildiz Technical University, Davutpasa Campus, 34210, Esenler, Istanbul, Turkey

    Aysegul Erdemir, Ebru Ozkan, Murat Topuzogullari, Zeynep Busra Bolat, Ayberk Akat & Dilek Turgut-Balik

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  1. Ebru Cayir
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  2. Aysegul Erdemir
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Correspondence to Dilek Turgut-Balik.

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Cayir, E., Erdemir, A., Ozkan, E. et al. Cloning of Intron-Removed Enolase Gene and Expression, Purification, Kinetic Characterization of the Enzyme from Theileria annulata . Mol Biotechnol 56, 689–696 (2014). https://doi.org/10.1007/s12033-014-9747-z

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  • DOI: https://doi.org/10.1007/s12033-014-9747-z

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