The c1 repressor of bacteriophage P1. Isolation and characterization of the repressor protein

Abstract

The c1 repressor gene of bacteriophage P1 is located on P1 DNA EcoRI fragment 7 (Sternberg, N. (1979) Virology 96, 129-142). Subfragments of P1 DNA EcoRI fragment 7 were cloned into expression vectors, and the c1 repressor protein from P1 wild-type phage and a revertant of a temperature-sensitive repressor mutant were overproduced in Escherichia coli and purified to near-homogeneity. The decreased electrophoretic mobility of P1 DNA BamHI fragment 9 in the presence of appropriate protein fractions was used as an assay for the repressor protein. Highly purified repressor migrates as a single polypeptide on denaturing sodium dodecyl sulfate-polyacrylamide gels, corresponding to a molecular weight of about 33,000. A molecular weight of about 63,000 for the native repressor molecule was calculated from determinations of the sedimentation coefficient, which was 2.6 s, and the Stokes radius, which was 55 A. Cross-linking the protein with glutaraldehyde yielded two bands. These data and a high frictional coefficient (2.1) suggest that the native repressor exists in solution as an asymmetric dimer molecule.