Isolation of the gal Repressor
- *Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014
- Laboratory of Biomedical Sciences, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014
- ‡Clinical Endocrinology Branch, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland 20014
Abstract
The repressor of the galactose operon of Escherichia coli has been partially purified and identified as a protein. Induction of a lysogen in which λ was linked to the bacterial galR and lysine genes resulted in a large increase in the production of the gal repressor. Single-step purification by affinity chromatography, using the ligand p-aminophenyl-β-D-thiogalactoside linked to beaded agarose, provided a convenient method of separating the gal repressor from other DNA-binding proteins. Binding of gal repressor to λpgal[32P]DNA was studied by assay of binding to a nitrocellulose filter. Interaction between gal repressor and λpgal DNA showed a high degree of specificity; the dissociation constant of the complex was estimated to be 1.0 × 10-12 M. Unlabeled λpgal DNA competed for binding to gal repressor, but λDNA and λh80dlac DNA did not. Fucose and galactose, which function as inducers of the galactose operon in vivo, produced one-half maximal inhibition of gal repressor-λpgal DNA binding at concentrations of 5 × 10-5 M.
