Optimized in vitro and in vivo expression of proteorhodopsin: A seven-transmembrane proton pump
Artikel i vetenskaplig tidskrift, 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.

Membrane protein

Cell-free

Proteorhodopsin

Functional expression

Författare

Pontus Emanuel Gourdon

Chalmers, Kemi- och bioteknik, Molekylär mikroskopi

Anna Alfredsson

Göteborgs universitet

Anders Pedersen

Göteborgs universitet

Erik Malmerberg

Göteborgs universitet

Anna Maria Nyblom

Chalmers, Kemi- och bioteknik, Molekylär mikroskopi

Mikael Widell

Chalmers University of Technology

Ronnie Berntsson

Rijksuniversiteit Groningen

Chalmers University of Technology

Jarone Pinhassi

Hogskolan i Kalmar

Marc Braiman

Syracuse University

Örjan Hansson

Göteborgs universitet

Nicklas Bonander

Göteborgs universitet

B Göran Karlsson

Göteborgs universitet

Richard Neutze

Göteborgs universitet

Protein Expression and Purification

1046-5928 (ISSN) 1096-0279 (eISSN)

Vol. 58 103-113

Ämneskategorier

Kemi

DOI

10.1016/j.pep.2007.10.017