Maximizing gene expression from plasmid vectors containing the lambda PL promoter: strategies for overproducing transcription termination factor rho.
We have constructed two plasmids in which transcription of the rho gene from Escherichia coli K-12 is under the control of the lambda phage PL promoter. 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 plasmids 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 approximately equal to 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. In contrast to chemical induction, heat induction in four cryptic lambda lysogens carrying the thermolabile cI857 repressor failed to yield the same high levels of rho protein (with either plasmid). Our results show that chemical induction of PL-containing plasmid expression vectors can serve as a convenient and useful alternative to the commonly used method of heat induction.
Mott
J E
JE
Grant
R A
RA
Ho
Y S
YS
Platt
T
T
D010582:Bacteriophage lambda / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D006358:Hot Temperature; D016685:Mitomycin; D008937:Mitomycins / Q000494:pharmacology; D009268:Nalidixic Acid / Q000494:pharmacology; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012234:Rho Factor / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D014157:Transcription Factors / Q000235:genetics*
D008937:Mitomycins; D012234:Rho Factor; D014157:Transcription Factors; D009268:Nalidixic Acid; D016685:Mitomycin