Open AccessExpression and characterisation of thekorBgene product from theStreptomyces lividansplasmid plJ101 inEscherichia coliand determination of its binding site on thekorBandkilBpromoters
Author(s) -
Sara Zaman,
Hilary Richards,
John M. Ward
Publication year - 1992
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/20.14.3693
Subject(s) - biology , microbiology and biotechnology , plasmid , gene , lac operon , transcription (linguistics) , escherichia coli , nucleic acid sequence , gene product , dna , streptomycetaceae , streptomyces , biochemistry , gene expression , genetics , actinomycetales , bacteria , linguistics , philosophy
A 0.5kb Spel-BclI fragment containing the pIJ101 korB ORF was cloned into pUC8 under the control of the lacZ promoter, creating pQR206. In vitro coupled transcription-translation of pQR206 identified a protein product of approximately 10kDa, which corresponds to the predicted molecular weight deduced from the korB sequence. pQR206 was used to express the 10kDa KorB protein in vivo in E. coli. Crude E. coli protein extracts containing KorB were shown to bind to a 0.8kb kilB fragment and a 0.5kb korB fragment in gel retardation assays. DNasel footprinting indicated that the DNA recognition sequence of the KorB protein lies within a 60bp protected region encompassing the kilB promoter and a 36bp region encompassing the korB promoter.