Overexpression, purification and characterization of the Escherichia coli MelR transcription activator protein

Abstract

The gene encoding Escherichia coli MelR protein has been cloned in the expression vector pJLA502. MelR has been overexpressed, substantially purified and shown to bind to DNA fragments carrying the melAB promoter. A truncated version of the melR gene, encoding the C-terminal half of MelR, was also cloned into pJLA502; the protein product of this truncated gene binds to the melAB promoter but was not overproduced. A number of amino acid substitutions were made in the recognition helices of two putative helix-turn-helix motifs in the C-terminal part of MelR, and the effects of these mutations on MelR-dependent transcription initiation at the melAB promoter have been measured.