Optimized in vitro and in vivo expression of proteorhodopsin : a seven-transmembrane proton pump
(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.
(Less)
- author
- publishing date
- 2008-03
- type
- Contribution to journal
- publication status
- published
- 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
-
- pmid:18191413
- scopus:38349172101
- ISSN
- 1046-5928
- DOI
- 10.1016/j.pep.2007.10.017
- language
- English
- LU publication?
- no
- id
- 536fa328-9c48-4dd2-aa3c-6ee64196e627
- date added to LUP
- 2017-04-29 15:32:15
- date last changed
- 2024-02-12 17:54:14
@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}}, 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}}, 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}}, doi = {{10.1016/j.pep.2007.10.017}}, volume = {{58}}, year = {{2008}}, }