Enhanced expression of membrane proteins in E. coli with a P(BAD) promoter mutant: synergies with chaperone pathway engineering strategies.

ABSTRACT: BACKGROUND: Membrane proteins (MPs) populate 20-30% of genomes sequenced to date and hold potential as therapeutic targets as well as for practical applications in bionanotechnology. However, MP toxicity and low yields in normally robust expression hosts such as E. coli has curtailed progress in our understanding of their structure and function. RESULTS: Using the seven transmembrane segments H. turkmenica deltarhodopsin (HtdR) as a reporter, we isolated a spontaneous mutant in the arabinose-inducible P(BAD) promoter leading to improved cell growth and a twofold increase in the recovery of active HtdR at 37°C. A single transversion in a conserved region of the cyclic AMP receptor protein binding site caused the phenotype by reducing htdR transcript levels by 65%. When the mutant promoter was used in conjunction with a host lacking the molecular chaperone Trigger Factor (?tig cells), toxicity was further suppressed and the amount of correctly folded HtdR was 4-fold that present in the membranes of control cells. More importantly, while improved growth barely compensated for the reduction in transcription rates when another polytopic membrane protein (N. pharonis sensory rhodopsin II) was expressed under control of the mutant promoter in wild type cells, a 4-fold increase in productivity could be achieved in a ?tig host. CONCLUSIONS: Our system, which combines a downregulated version of the tightly repressed P(BAD) promoter with a TF-deficient host may prove a valuable alternative to T7-based expression for the production of membrane proteins that have so far remained elusive targets.

SUBMITTER: Nannenga BL 

PROVIDER: S-EPMC3265434 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature