title: Cloning, overexpression, purification, and matrix-assisted refolding of DevS (Rv 3132c) histidine protein kinase of Mycobacterium tuberculosis.
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.
authors.affiliation: Department of Biotechnology, All India Institute of Medical Sciences, New Delhi 110029, India.
authors.forename: Deepak Kumar
authors.initials: DK
authors.lastname: Saini
authors.forename: Neha
authors.initials: N
authors.lastname: Pant
authors.forename: Taposh K
authors.initials: TK
authors.lastname: Das
authors.forename: Jaya Sivaswami
authors.initials: JS
authors.lastname: Tyagi
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002384:Catalysis; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D000071677:Histidine Kinase; D002479:Inclusion Bodies / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D008957:Models, Genetic; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010766:Phosphorylation; D011487:Protein Conformation; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D011817:Rabbits; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C098956:DevR protein, Nostoc; D014157:Transcription Factors; D011494:Protein Kinases; D000071677:Histidine Kinase