Purification and characterization of trp aporepressor.

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Abstract
Homogeneous trp aporepressor was isolated from an overproducing strain of E. 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 RsaI 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 2 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, 1 dimer is bound to the operator site with an apparent dissociation constant < 2 .times. 10-9 M. Preliminary equilibrium dialysis experiments suggest that tryptophan binds to the aporepressor with a dissociation constant of 1.6 .times. 10-5 M.