Maximizing gene expression from plasmid vectors containing the λ P(L) promoter: Strategies for overproducing transcription termination factor ρ

Abstract

We have constructed two plasmids in which transcription of the ρ gene from Escherichia coli K-12 is under the control of the λ phage P(L) promoter. In p31-356, the normal ρ promoter is deleted, but the remainder of the ρ leader region, including the ribosome binding site, is present. In p39-AS, the ρ leader is completely absent, and the λ cII ribosome binding site replaces that of ρ. Under noninducing conditions, expression of ρ protein from these plasmids is repressed by the λ cI protein in hosts carrying λ cryptic prophage. Induction using mitomycin C or nalidixic acid in a cryptic lysogen carrying the cI+ repressor resulted in the overproduction of ρ protein to levels of 3%-5% of the total cellular protein with p31-356, and to levels of ~40% with p39-AS. The overproduced protein is functionally indistinguishable from the ρ protein isolated from the K-12 strain W3110, and it can be obtained from cells harboring p39-As in yields of up to 25 mg of ρ per g of cells. In contrast to chemical induction, heat induction in four cryptic λ lysogens carrying the thermolabile cI857 repressor failed to yield the same high levels of ρ protein (with either plasmid). Our results show that chemical induction of P(L)-containing plasmid expression vectors can serve as a convenient and useful alternative to the commonly used method of heat induction.