Maximizing gene expression from plasmid vectors containing the lambda PL promoter: strategies for overproducing transcription termination factor rho.

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Abstract
Two plasmids in which transcription of the .rho. gene from Escherichia coli K-12 is under the control of the .lambda. phage PL promoter were constructed. In p31-356, the normal .rho. promoter is deleted, but the remainder of the .rho. leader region, including the ribosome binding site, is present. In p39-AS, the .rho. leader is completely absent and the .lambda. cII ribosome binding site replaces that of .rho.. Under noninducing conditions, expression of .rho. protein from these plamids is repressed by the .lambda. cI protein in hosts carrying .lambda. cryptic prophage. Induction using mitomycin C or nalidixic acid in a cryptic lysogen carrying the cI+ repressor resulted in the overproduction of .rho. protein to levels of 3-5% of the total cellular protein with p31-356 and to levels of .apprxeq. 40% with p39-AS. The overproduced protein is functionally indistinguishable from the .rho. 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 .rho. per g of cells. Heat induction in 4 cryptic .lambda. lysogens carrying the thermolabile cI857 repressor failed to yield the same high levels of .rho. protein (with either plasmid). Chemical induction of PL containing plasmid expression vectors can serve as a convenient and useful alternative to the commonly used method of heat induction.