In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals.
We report the construction and use of a series of plasmid vectors suitable for the detection and cloning of translational control signals and 5' coding sequences of exogenously derived genes. In these plasmids, the first eight codons of the amino-terminal end of the lactose operon beta-galactosidase gene, lacZ, were removed, and unique BamHI, EcoRI, and SmaI (XmaI) endonuclease cleavage sites were incorporated adjacent to the eighth codon of lacZ. Introduction of deoxyribonucleic acid fragments containing appropriate regulatory signals and 5' coding sequences into such lac fusion plasmids led to the production of hybrid proteins consisting of the carboxyl-terminal segment of a beta-galactosidase remnant plus a peptide fragment that contained the amino-terminal amino acids encoded by the exogenous deoxyribonucleic acid sequence. These hybrid peptides retained beta-galactosidase enzymatic activity and yielded a Lac+ phenotype. Such hybrid proteins are useful for purifying peptide sequences encoded by exogenous deoxyribonucleic acid fragments and for studies relating the structure and function of specific peptide segments.
D001483:Base Sequence; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D005696:Galactosidases / Q000235:genetics*; D007763:Lac Operon; D010957:Plasmids; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
D004274:DNA, Recombinant; D012329:RNA, Bacterial; D012333:RNA, Messenger; D005696:Galactosidases; D001616:beta-Galactosidase