Optimized in vitro and in vivo expression of proteorhodopsin: a seven-transmembrane proton pump

Protein Expr Purif. 2008 Mar;58(1):103-13. doi: 10.1016/j.pep.2007.10.017. Epub 2007 Nov 20.

Abstract

Proteorhodopsin is an integral membrane light-harvesting proton pump that is found in bacteria distributed throughout global surface waters. Here, we present a protocol for functional in vitro production of pR using a commercial cell-free synthesis system yielding 1.0mg purified protein per milliliter of cell lysate. We also present an optimized protocol for in vivo over-expression of pR in Escherichia coli, and a two-step purification yielding 5mg of essentially pure functional protein per liter of culture. Both approaches are straightforward, rapid, and easily scalable. Thus either may facilitate the exploitation of pR for commercial biotechnological applications. Finally, the implications of some observations of the in vitro synthesis behavior, as well as preliminary results towards a structural determination of pR are discussed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bioreactors
  • Cloning, Molecular
  • Gammaproteobacteria / genetics
  • Gammaproteobacteria / metabolism
  • Gene Expression
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Sorting Signals
  • Proton Pumps
  • Rhodopsin* / biosynthesis
  • Rhodopsin* / chemistry
  • Rhodopsin* / genetics
  • Rhodopsin* / isolation & purification
  • Rhodopsins, Microbial

Substances

  • Protein Sorting Signals
  • Proton Pumps
  • Rhodopsins, Microbial
  • proteorhodopsin
  • Rhodopsin