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 beta-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 1-1 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.
Bishop
R E
RE
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Weiner
J H
JH
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D016971:Citrobacter freundii / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D015870:Gene Expression; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012995:Solubility; D014157:Transcription Factors / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D014157:Transcription Factors; C071526:AmpR protein, Bacteria