Optimized in vitro and in vivo expression of proteorhodopsin: A seven-transmembrane proton pump
Journal article, 2008
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 1.0 mg 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 5 mg 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.
Proteorhodopsin
Functional expression
Cell-free
Membrane protein
Author
Pontus Emanuel Gourdon
Chalmers, Chemical and Biological Engineering, Molecular Imaging
Anna Alfredsson
University of Gothenburg
Anders Pedersen
University of Gothenburg
Erik Malmerberg
University of Gothenburg
Anna Maria Nyblom
Chalmers, Chemical and Biological Engineering, Molecular Imaging
Mikael Widell
Chalmers University of Technology
Ronnie Berntsson
Chalmers University of Technology
University of Groningen
Jarone Pinhassi
Hogskolan i Kalmar
Marc Braiman
Syracuse University
Örjan Hansson
University of Gothenburg
Nicklas Bonander
University of Gothenburg
B Göran Karlsson
University of Gothenburg
Richard Neutze
University of Gothenburg
Protein Expression and Purification
1046-5928 (ISSN) 1096-0279 (eISSN)
Vol. 58 1 103-113Subject Categories (SSIF 2011)
Chemical Sciences
DOI
10.1016/j.pep.2007.10.017