Purification and characterization of trp aporepressor

Abstract

We have isolated homogeneous trp aporepressor from an overproducing strain of Escherichia coli carrying a plasmid containing trpR preceded by tandem trp operon promoters. Dye-affinity and ion-exchange chromatography were used in conjunction with a gel electrophoresis assay in which the repressor, when bound to the trp operator, protects an Rsa I restriction site from endonuclease cleavage. Crystals suitable for x-ray diffraction studies were grown from a variety of concentrated salt solutions. Hydrodynamic properties and electrophoretic analysis of unmodified and covalently crosslinked aporepressor show that the free aporepressor has an isoelectric point of 5.9 and is a dimer containing two identical 12.5-kilodalton subunits in the presence or absence of L-tryptophan. The repressor . operator complex binds poorly to nitrocellulose filters, but restriction-site protection studies indicate that, in the presence of tryptophan, one dimer is bound to the operator site with an apparent dissociation constant less than 2 X 10(-9) M. Preliminary equilibrium dialysis experiments suggest that tryptophan binds to the aporepressor with a dissociation constant of 1.6 X 10(-5) M.