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

Pontus Gourdon (Institutionen för kemi- och bioteknik, Molekylär mikroskopi) ; Anna Alfredsson ; Anders Pedersen ; Erik Malmerberg ; Maria Nyblom (Institutionen för kemi- och bioteknik, Molekylär mikroskopi) ; Mikael Widell ; Ronnie Berntsson ; Jarone Pinhassi ; Marc Braiman ; Örjan Hansson ; Nicklas Bonander ; B Göran Karlsson ; Richard Neutze
Protein Expression and Purification Vol. 58 (2008), 1, p. 103-113.
[Artikel, refereegranskad vetenskaplig]

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.

Nyckelord: Proteorhodopsin; Membrane protein; Cell-free; Functional expression



Denna post skapades 2008-10-30. Senast ändrad 2011-01-20.
CPL Pubid: 76650

 

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Institutioner (Chalmers)

Institutionen för kemi- och bioteknik, Molekylär mikroskopi (2008-2014)
Institutionen för kemi (2001-2011)
Svenskt NMR-centrum vid Göteborgs universitet (GU)

Ämnesområden

Kemi

Chalmers infrastruktur