Reference - Detail
|Author||Mujacic M, Cooper KW, Baneyx F.|
|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.|
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.
|MeSH||Bacterial Proteins / genetics* Base Sequence Cloning, Molecular Cold Temperature DNA Primers Escherichia coli / genetics* Escherichia coli Proteins* Genetic Vectors* Hydrolysis Promoter Regions, Genetic Recombinant Fusion Proteins / genetics* Recombinant Fusion Proteins / metabolism Recombinant Fusion Proteins / toxicity Transcription, Genetic / genetics beta-Lactamases / genetics*|
|WOS Category||GENETICS & HEREDITY|
|Prokaryotes E. coli||pCS22 pCS24|