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

Brent L. Nannenga, François Baneyx

Research output: Contribution to journalArticle

13 Scopus citations

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 (Δtig 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 Δtig 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.

Original languageEnglish (US)
Article number105
JournalMicrobial cell factories
Volume10
DOIs
StatePublished - Dec 9 2011
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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