Expression vector with two-step control by the cI-pR-Q-p′R-qut-t′R module of coliphage lambda

A plasmid expression vector (pCEQ3), using temperature-regulated transcription from the p'R promoter of bacteriophage lambda, has been constructed. The vector is derived from pBR327 in which the EcoRI-ClaI fragment of plasmid DNA is replaced with a 2.2-kb DNA module cI857-pR-Q-p'R-qut-t'R, consisting of two regions of the lambda genome. The first region contains the repressor gene cI857 and promoter pR; the second one contains gene Q and the late promoter p'R. When the repressor protein, product of the cI857 gene, becomes temperature-inactivated, it allows the promoter pR to initiate the transcription of the Q gene. The product of the Q gene, in turn, acts as a positive regulator of transcription from promoter p'R. The promoter activity of pR is fully repressed at a low temperature (30 degrees C) and transcription from p'R is terminated in the absence of Q gene product, but the shift of temperature up to 37 degrees C is sufficient to make the transcription from the p'R promoter highly active. Foreign genes can be inserted into the single EcoRI site downstream from the p'R promoter. The resultant constructions express extremely high levels of the cloned gene product in Escherichia coli.