Cold-inducible cloning vectors for low-temperature protein expression in Escherichia coli: application to the production of a toxic and proteolytically sensitive fusion protein

TolAI-β-lactamase a fusion protein consisting of the inner membrane anchoring domain of the Escherichia coli transenvelope protein TolA followed by TEM-β-lactamase was found to be toxic and highly unstable when transcribed from the bacteriophage T7 promoter at 37°C. Expression at 15 or 23°C alleviated toxicity, but led to only partial stabilization of the fusion protein. To evaluate the usefulness of cold-shock promoters for the production of proteolytically sensitive proteins at low temperatures, we constructed a set of cloning vectors suitable for rapidly positioning PCR products under cspA transcriptional control. TolAI-β-lactamase degradation was completely abolished when cspA-driven transcription was induced by temperature downshift to 15 or 23°C. Our results suggest that the cspA promoter system may be a valuable tool for the production of proteins containing membrane-spanning domains or otherwise unstable gene products in E. coli.