beta-Galactosidase chimeras: primary structure of a lac repressor-beta-galactosidase protein.

Abstract

A protein possessing both lac repressor and .beta.-galactosidase activities in a single polypeptide of about 155,000 daltons was purified from a deletion mutant of Escherichia coli in which the lacI and Z genes are fused. A 77-residue CNBr peptide containing the fusion joint was isolated. A radioimmunoassay with an antibody prepared against CNBr2 (residues 3-92) of .beta.-galactosidase was used to monitor its purification. The sequence of the joining peptide was determined by analysis of tryptic peptides and by automatic sequencer analysis. The site of joining is from residue 355 of lac repressor to residue 24 of .beta.-galactosidase (or 356 to 25), indicating that the last 4 residues at the carboxyl terminus of lac repressor and the first 23 residues at the amino terminus of .beta.-galactosidase are not essential for the activities of these 2 proteins. The exact site of the fusion is not known because lac repressor residue 356 and .beta.-galactosidase residue 24 are both leucine residues. Examination of the nucleotide sequences around the 2 end points of the deletion revealed a homology of 9 identities in a stretch of 11 base pairs.