title: 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 (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.
authors.affiliation: Department of Chemical Engineering, University of Washington, Seattle, WA 98195-1750, USA.
authors.forename: Brent L
authors.initials: BL
authors.lastname: Nannenga
authors.forename: François
authors.initials: F
authors.lastname: Baneyx
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006216:Halobacteriaceae / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering; D018832:Molecular Chaperones / Q000235:genetics* / Q000378:metabolism; D009876:Operon; D019696:Peptidylprolyl Isomerase / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D020218:Response Elements; D012243:Rhodopsin / Q000235:genetics* / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D012243:Rhodopsin; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase