Parks J S, Gottesman M, Shimada K, Weisberg R A, Perlman R L, Pastan I
Proc Natl Acad Sci U S A. 1971 Aug;68(8):1891-5. doi: 10.1073/pnas.68.8.1891.
The repressor of the galactose operon of Escherichia coli has been partially purified and identified as a protein. Induction of a lysogen in which lambda 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-beta-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 lambdapgal[(32)P]DNA was studied by assay of binding to a nitrocellulose filter. Interaction between gal repressor and lambdapgal DNA showed a high degree of specificity; the dissociation constant of the complex was estimated to be 1.0 x 10(-12) M. Unlabeled lambdapgal DNA competed for binding to gal repressor, but lambdaDNA and lambdah80dlac DNA did not. Fucose and galactose, which function as inducers of the galactose operon in vivo, produced one-half maximal inhibition of gal repressor-lambdapgal DNA binding at concentrations of 5 x 10(-5) M.
大肠杆菌半乳糖操纵子的阻遏物已被部分纯化并鉴定为一种蛋白质。诱导λ噬菌体与细菌galR和赖氨酸基因相连的溶原菌，会导致半乳糖阻遏物的产量大幅增加。使用与琼脂糖珠相连的配体对氨基苯基-β-D-硫代半乳糖苷通过亲和层析进行一步纯化，提供了一种将半乳糖阻遏物与其他DNA结合蛋白分离的便捷方法。通过检测与硝酸纤维素滤膜的结合来研究半乳糖阻遏物与λpgal[(32)P]DNA的结合。半乳糖阻遏物与λpgal DNA之间的相互作用表现出高度的特异性；复合物的解离常数估计为1.0×10(-12)M。未标记的λpgal DNA竞争与半乳糖阻遏物的结合，但λDNA和λh80dlac DNA则不然。在体内作为半乳糖操纵子诱导剂的岩藻糖和半乳糖，在浓度为5×10(-5)M时对半乳糖阻遏物-λpgal DNA结合产生最大抑制作用的一半。
