Isolation of a set of hybrid lac repressors made in vitro between normal lac repressor and its homogeneous tryptic core.

Abstract

Lactose repressor can be renatured from 8 M guanidine-HCl solution. The renatured repressor is tetrameric and shows DNA binding activity. Thus it becomes possible to obtain hybrid tetramers in vitro between normal repressor and repressor defective in DNA binding by simultaneous denaturation and renaturation. In order to facilitate the separation of the different hybrids, we have used a lac repressor derivative that does not bind DNA, which is missing the amino-terminal 59 residues of the polypeptide chain (homogeneous tryptic core). The hybrids resulting from the mixed renaturation of homogeneous tryptic core and normal repressor can be separated by electrophoresis on Cellogel. The hybrids have been recovered, and a preliminary characterization of their DNA-binding properties is reported.