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

Gourdon, Pontus LU ; Alfredsson, Anna; Pedersen, Anders G.; Malmerberg, Erik; Nyblom, Maria; Widell, Mikael; Berntsson, Ronnie; Pinhassi, Jarone; Braiman, Marc and Hansson, Orjan, et al. (2008) In Protein Expression and Purification 58(1). p.13-103
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... (More)

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.

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keywords
Bioreactors, Cloning, Molecular, Gammaproteobacteria, Gene Expression, Nuclear Magnetic Resonance, Biomolecular, Protein Sorting Signals, Proton Pumps, Rhodopsin, Rhodopsins, Microbial, Journal Article, Research Support, Non-U.S. Gov't
in
Protein Expression and Purification
volume
58
issue
1
pages
13 - 103
publisher
Academic Press
external identifiers
  • scopus:38349172101
ISSN
1046-5928
DOI
10.1016/j.pep.2007.10.017
language
English
LU publication?
no
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536fa328-9c48-4dd2-aa3c-6ee64196e627
date added to LUP
2017-04-29 15:32:15
date last changed
2017-05-07 04:52:51
@article{536fa328-9c48-4dd2-aa3c-6ee64196e627,
  abstract     = {<p>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.</p>},
  author       = {Gourdon, Pontus and Alfredsson, Anna and Pedersen, Anders G. and Malmerberg, Erik and Nyblom, Maria and Widell, Mikael and Berntsson, Ronnie and Pinhassi, Jarone and Braiman, Marc and Hansson, Orjan and Bonander, Nicklas and Karlsson, Göran and Neutze, Richard},
  issn         = {1046-5928},
  keyword      = {Bioreactors,Cloning, Molecular,Gammaproteobacteria,Gene Expression,Nuclear Magnetic Resonance, Biomolecular,Protein Sorting Signals,Proton Pumps,Rhodopsin,Rhodopsins, Microbial,Journal Article,Research Support, Non-U.S. Gov't},
  language     = {eng},
  number       = {1},
  pages        = {13--103},
  publisher    = {Academic Press},
  series       = {Protein Expression and Purification},
  title        = {Optimized in vitro and in vivo expression of proteorhodopsin : a seven-transmembrane proton pump},
  url          = {http://dx.doi.org/10.1016/j.pep.2007.10.017},
  volume       = {58},
  year         = {2008},
}