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- Schwarz, Daniel, et al.
(författare)
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Cell-free expression profiling of E. coli inner membrane proteins
- 2010
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 10:9, s. 1762-1779
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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