title: Cold-inducible cloning vectors for low-temperature protein expression in Escherichia coli: application to the production of a toxic and proteolytically sensitive fusion protein.
abstract: TolAI-beta-lactamase a fusion protein consisting of the inner membrane anchoring domain of the Escherichia coli transenvelope protein TolA followed by TEM-beta-lactamase was found to be toxic and highly unstable when transcribed from the bacteriophage T7 promoter at 37 degrees C. Expression at 15 or 23 degrees 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-beta-lactamase degradation was completely abolished when cspA-driven transcription was induced by temperature downshift to 15 or 23 degrees 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.
authors.affiliation: Department of Chemical Engineering, University of Washington, Seattle 98195-1750, USA.
authors.forename: M
authors.initials: M
authors.lastname: Mujacic
authors.forename: K W
authors.initials: KW
authors.lastname: Cooper
authors.forename: F
authors.initials: F
authors.lastname: Baneyx
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003080:Cold Temperature; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D006868:Hydrolysis; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D014158:Transcription, Genetic / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C062260:cold shock protein CS7.4, Bacteria; C062093:tolA protein, E coli; D001618:beta-Lactamases