Cloning, overexpression, purification, and matrix-assisted refolding of DevS (Rv 3132c) histidine protein kinase of Mycobacterium tuberculosis.
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.
Saini
Deepak Kumar
DK
Department of Biotechnology, All India Institute of Medical Sciences, New Delhi 110029, India.
Pant
Neha
N
Das
Taposh K
TK
Tyagi
Jaya Sivaswami
JS
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
D001426:Bacterial Proteins; C098956:DevR protein, Nostoc; D014157:Transcription Factors; D011494:Protein Kinases; D000071677:Histidine Kinase