title: Optimized in vitro and in vivo expression of proteorhodopsin: a seven-transmembrane proton pump.
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.
authors.affiliation: Department of Chemical and Biological Engineering, Molecular Biotechnology, Chalmers University of Technology, P.O. Box 462, SE-40530 Göteborg, Sweden.
authors.forename: Pontus
authors.initials: P
authors.lastname: Gourdon
authors.forename: Anna
authors.initials: A
authors.lastname: Alfredsson
authors.forename: Anders
authors.initials: A
authors.lastname: Pedersen
authors.forename: Erik
authors.initials: E
authors.lastname: Malmerberg
authors.forename: Maria
authors.initials: M
authors.lastname: Nyblom
authors.forename: Mikael
authors.initials: M
authors.lastname: Widell
authors.forename: Ronnie
authors.initials: R
authors.lastname: Berntsson
authors.forename: Jarone
authors.initials: J
authors.lastname: Pinhassi
authors.forename: Marc
authors.initials: M
authors.lastname: Braiman
authors.forename: Orjan
authors.initials: O
authors.lastname: Hansson
authors.forename: Nicklas
authors.initials: N
authors.lastname: Bonander
authors.forename: Göran
authors.initials: G
authors.lastname: Karlsson
authors.forename: Richard
authors.initials: R
authors.lastname: Neutze
mesh_terms: D019149:Bioreactors; D003001:Cloning, Molecular; D020563:Gammaproteobacteria / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D019906:Nuclear Magnetic Resonance, Biomolecular; D021382:Protein Sorting Signals; D017494:Proton Pumps; D012243:Rhodopsin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D025604:Rhodopsins, Microbial
chemical_list: D021382:Protein Sorting Signals; D017494:Proton Pumps; D025604:Rhodopsins, Microbial; C413548:proteorhodopsin; D012243:Rhodopsin