EXPRESSION AND PURIFICATION OF THE CYNR REGULATORY GENE-PRODUCT - CYNR IS A DNA-BINDING PROTEIN

The CynR protein, a member of the LysR family, positively regulates th e Escherichia coli cyn operon and negatively autoregulates its own tra nscription. By Sl mapping analysis, the in vivo cynR transcription sta rt site was located 63 bp upstream of the cyn TSX operon transcription start site. Topologically, the cynR and cynTSX promoters overlap and direct transcription in opposite directions. The CynR translation init iation codon was identified by oligonucleotide-directed mutagenesis, a nd the CynR coding sequence was cloned under the control of a T7 phage promoter. The CynR protein was stably expressed at a high level with a T7 RNA polymerase-T7 phage promoter system. Purification by ion-exch ange chromatography, affinity chromatography, and ammonium sulfate fra ctionation yielded pure CynR protein. Gel shift assays confirmed that CynR is a DNA-binding protein like the other members of the LysR famil y. The CynR regulatory protein binds specifically to a 136-bp DNA frag ment encompassing both the cynR and the cynTSX promoters.