Open AccessIsolation of the gal Repressor
Author(s) -
John S. Parks,
M. Gottesman,
Kazunori Shimada,
Robert A. Weisberg,
Robert L. Perlman,
Ira Pastan
Publication year - 1971
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.68.8.1891
Subject(s) - isolation (microbiology) , repressor , computational biology , biology , chemistry , genetics , microbiology and biotechnology , gene , transcription factor
The repressor of the galactose operon ofEscherichia coli has been partially purified and identified as a protein. Induction of a lysogen in which λ was linked to the bacterialgal R and lysine genes resulted in a large increase in the production of thegal repressor. Single-step purification by affinity chromatography, using the ligandp -aminophenyl-β-D-thiogalactoside linked to beaded agarose, provided a convenient method of separating thegal repressor from other DNA-binding proteins. Binding ofgal repressor to λpgal [32 P]DNA was studied by assay of binding to a nitrocellulose filter. Interaction betweengal 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 togal repressor, but λDNA and λh80dlac DNA did not. Fucose and galactose, which function as inducers of the galactose operonin vivo , produced one-half maximal inhibition ofgal repressor-λpgal DNA binding at concentrations of 5 × 10-5 M.