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In Vitro Refolding of Triosephosphate Isomerase from L. donovani

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Abstract

The triosephosphate isomerase of Leishmania donovani (LdTIM) was expressed at high level in Escherichia coli. The TIM gene was cloned in expression vector pET-23(a) with C-terminal 6× His tag fused in frame, and expressed as a 27.6-kDa protein in E. coli as inclusion bodies. The recombinant LdTIM from E. coli lysate was solubilized in 6 M guanidine hydrochloride and purified by Ni-NTA chromatography. In the present study, the effect of bovine serum albumin on the reactivation of TIM was investigated. Furthermore, 8-anilino-1-naphthalene sulfonic acid was used to detect the structural changes induced by bovine serum albumin (BSA). Here, we conclude that BSA assists in the refolding and regain of LdTIM enzyme activity by providing framework for structure formation. This study indicates that numerous protein–protein contacts are constantly occurring inside the cell that leads to the formation of native protein.

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Acknowledgments

We thankfully acknowledge Dr. Tushar Kanti Chakraborty for constant support provided during the studies. Kishore Kumar thanks Council of Scientific and Industrial Research, New Delhi, India for providing Senior Research Fellowship. CDRI communication is 145/2010/UR.

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  1. Division of Biochemistry, Central Drug Research Institute, Lucknow, 226001, UP, India

    Kishore Kumar, Prachi Bhargava & Uma Roy

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  1. Kishore Kumar
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  2. Prachi Bhargava
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  3. Uma Roy
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Correspondence to Uma Roy.

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Kumar, K., Bhargava, P. & Roy, U. In Vitro Refolding of Triosephosphate Isomerase from L. donovani . Appl Biochem Biotechnol 164, 1207–1214 (2011). https://doi.org/10.1007/s12010-011-9206-2

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  • DOI: https://doi.org/10.1007/s12010-011-9206-2

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