Cloning, Overexpression, Purification, and Matrix-Assisted Refolding of DevS (Rv 3132c) Histidine Protein Kinase of Mycobacterium tuberculosis

Authors: Saini D.K.¹; Pant N.¹; Das T.K.²; Tyagi J.S.¹

Source: Protein Expression and Purification, Volume 25, Number 1, June 2002 , pp. 203-208(6)

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Abstract:

The devR-devS (Rv 3133c-Rv 3132c) two-component system of Mycobacterium tuberculosis was identified in our laboratory by RNA subtractive hybridization. This genetic system was predicted to encode a response regulator and histidine protein kinase, respectively. The putative histidine kinase protein DevS was overexpressed to high levels in Escherichia coli as a fusion protein with a hexahistidine tag, His(6)-DevS₂₀₁, in the form of inclusion bodies. Here we report a “redox-based” method of matrix-bound renaturation of DevS protein. The refolded protein was biochemically active in an autophosphorylation reaction characteristic of histidine kinases and was suitable for the generation of polyclonal antibodies and as an antigen in ELISA. Copyright 2002 Elsevier Science (USA).

Keywords: Mycobacterium tuberculosis; two-component system; sensor kinase; poly-His tag; inclusion bodies; protein renaturation.

Language: English

Document Type: Research article

Affiliations: 1: Department of Biotechnology 2: Department of Anatomy, All India Institute of Medical Sciences, New Delhi, 110029, India

Publication date: 2002-06-01