Optimized in vitro and in vivo expression of proteorhodopsin: a seven-transmembrane proton pump.
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.
Gourdon
Pontus
P
Department of Chemical and Biological Engineering, Molecular Biotechnology, Chalmers University of Technology, P.O. Box 462, SE-40530 Göteborg, Sweden.
Alfredsson
Anna
A
Pedersen
Anders
A
Malmerberg
Erik
E
Nyblom
Maria
M
Widell
Mikael
M
Berntsson
Ronnie
R
Pinhassi
Jarone
J
Braiman
Marc
M
Hansson
Orjan
O
Bonander
Nicklas
N
Karlsson
Göran
G
Neutze
Richard
R
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
D021382:Protein Sorting Signals; D017494:Proton Pumps; D025604:Rhodopsins, Microbial; C413548:proteorhodopsin; D012243:Rhodopsin