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Cell-free expression profiling of E. coli inner membrane proteins
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2010 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 10, no 9, 1762-1779 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2010. Vol. 10, no 9, 1762-1779 p.
Keyword [en]
Detergent solubilization, Flavocytochrome, Green fluorescent protein, Inner membrane proteome, Technology, Transport proteins
National Category
Natural Sciences
URN: urn:nbn:se:su:diva-50120DOI: 10.1002/pmic.200900485ISI: 000277730900004OAI: diva2:380617
authorCount :5Available from: 2010-12-21 Created: 2010-12-21 Last updated: 2010-12-21Bibliographically approved

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Daley, Daniel
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Department of Biochemistry and Biophysics
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