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A rapid expression and purification condition screening protocol for membrane protein structural biology
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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Number of Authors: 5
2017 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 26, no 8, 1653-1666 p.Article in journal (Refereed) Published
Abstract [en]

Membrane proteins control a large number of vital biological processes and are often medically important-not least as drug targets. However, membrane proteins are generally more difficult to work with than their globular counterparts, and as a consequence comparatively few high-resolution structures are available. In any membrane protein structure project, a lot of effort is usually spent on obtaining a pure and stable protein preparation. The process commonly involves the expression of several constructs and homologs, followed by extraction in various detergents. This is normally a time-consuming and highly iterative process since only one or a few conditions can be tested at a time. In this article, we describe a rapid screening protocol in a 96-well format that largely mimics standard membrane protein purification procedures, but eliminates the ultracentrifugation and membrane preparation steps. Moreover, we show that the results are robustly translatable to large-scale production of detergent-solubilized protein for structural studies. We have applied this protocol to 60 proteins from an E. coli membrane protein library, in order to find the optimal expression, solubilization and purification conditions for each protein. With guidance from the obtained screening data, we have also performed successful large-scale purifications of several of the proteins. The protocol provides a rapid, low cost solution to one of the major bottlenecks in structural biology, making membrane protein structures attainable even for the small laboratory.

Place, publisher, year, edition, pages
2017. Vol. 26, no 8, 1653-1666 p.
Keyword [en]
membrane protein, E. coli, FSEC, GFP, detergent screening, IMAC purification, structural biology
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:su:diva-145854DOI: 10.1002/pro.3196ISI: 000406123600018OAI: oai:DiVA.org:su-145854DiVA: diva2:1135607
Available from: 2017-08-23 Created: 2017-08-23 Last updated: 2017-08-23Bibliographically approved

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Sjöstrand, DanDiamanti, RiccardoLundgren, Camilla A. K.Wiseman, BenjaminHögbom, Martin
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