Overproduction, solubilization, purification and DNA‐binding properties of AmpR from Citrobacter freundii

AmpR belongs to the LysR family of prokaryotic DNA‐binding transcriptional regulators and controls induction of the enterobacterial ampC β‐lactamase gene. The ampR gene of Citrobacter freundii was deregulated by employing the polymerase chain reaction to introduce an efficient ribosome‐binding sequence and suitable restriction enzyme sites for cloning into a chemically inducible tac ‐promoter expression vector. When induced in Escherichia coli , the modified ampR gene rapidly overproduced the AmpR protein as an insoluble aggregate. The AmpR protein could be solubilized with 1.32 M guanidine/HCl and remained soluble when dialyzed against 0.5 M NaCl. The solubility properties of AmpR were exploited to selectively precipitate and resolubilize the protein in a nearly homogenous state. AmpR was then purified by a single gel‐filtration chromatography step which demonstrated that AmpR exists in solution as a monodisperse homodimeric protein. Several milligrams of purified AmpR could be obtained routinely from a 1‐l culture of induced bacteria. A DNA‐binding assay buffer containing 300 mM potassium glutamate and 30% glycerol was found to stabilize AmpR and used to demonstrate sequence‐specific DNA‐binding. Additionally, purified AmpR binds a half‐operator DNA with an inverted‐repeat sequence which competes with binding by the wild‐type operator. These findings are discussed in terms of the helix‐turn‐helix DNA‐binding motif, whereby AmpR is proposed to interact with its wild‐type operator as a dimer of dimers.