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Cell-free expressio...
Cell-free expression profiling of E. coli inner membrane proteins
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Schwarz, Daniel (författare)
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- Daley, Daniel (författare)
- Stockholms universitet,Institutionen för biokemi och biofysik
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Beckhaus, Tobias (författare)
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Doetsch, Volker (författare)
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Bernhard, Frank (författare)
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(creator_code:org_t)
- 2010-03-02
- 2010
- Engelska.
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 10:9, s. 1762-1779
- Relaterad länk:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- The high versatility and open nature of cell-free expression systems offers unique options to modify expression environments. In particular for membrane proteins, the choice of co-translational versus post-translational solubilization approaches could significantly modulate expression efficiencies and even sample qualities. The production of a selection of 134 a-helical integral membrane proteins of the Escherichia cob inner membrane proteome focussing on larger transporters has therefore been evaluated by a set of individual cell-free expression reactions. The production profiles of the targets in different cell-free expression modes were analyzed independently by three screening strategies. Translational green fluorescent protein fusions were analyzed as monitor for the formation of proteomicelles after cell-free expression of membrane proteins in the presence of detergents. In addition, two different reaction configurations were implemented and performed either by robotic semi-throughput approaches or by individually designed strategies. The expression profiles were specified for the particular cell-free modes and overall, the production of 87% of the target list could be verified and approximately 50% could already be synthesized in preparative scales. The expression of several selected targets was up-scaled to milliliter volumes and milligram amounts of production. As an example, the flavocytochrome YedZ was purified and its sample quality was demonstrated.
Nyckelord
- Detergent solubilization
- Flavocytochrome
- Green fluorescent protein
- Inner membrane proteome
- Technology
- Transport proteins
- NATURAL SCIENCES
- NATURVETENSKAP
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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