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Practical Aspects in Expression and Purification of Membrane Proteins for Structural Analysis

  • Kutti R. Vinothkumar
  • Patricia C. Edwards
  • Joerg Standfuss
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 955)

Abstract

A surge of membrane protein structures in the last few years can be attributed to advances in technologies starting at the level of genomes, to highly efficient expression systems, stabilizing conformational flexibility, automation of crystallization and data collection for screening large numbers of crystals and the microfocus beam lines at synchrotrons. The substantial medical importance of many membrane proteins provides a strong incentive to understand them at the molecular level. It is becoming obvious that the major bottleneck in many of the membrane projects is obtaining sufficient amount of stable functional proteins in a detergent micelle for structural studies. Naturally, large effort has been spent on optimizing and advancing multiple expression systems and purification strategies that have started to yield sufficient protein and structures. We describe in this chapter protocols to refold membrane proteins from inclusion bodies, purification from inner membranes of Escherichia coli and from mammalian cell lines.

Key words

Expression systems Escherichia coli Inclusion bodies HEK cells Rhodopsin 

Notes

Acknowledgments

VK acknowledges the European Union for a Marie-Curie Intra-European Fellowship and the Medical Research Council for a Career Development fellowship. The work on OmpG was done by VK at the Max-Planck Institute of Biophysics, Frankfurt as part of Ph.D. thesis. This work was further supported by the Swiss National Science Foundation (SNSF) grants 31003A_132815 and 31003A_141235 to JS. We thank Gebhard Schertler at the PSI and Daniel Oprian at the Brandeis University for discussion and advice on the expression of rhodopsin.

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Kutti R. Vinothkumar
    • 1
  • Patricia C. Edwards
    • 1
  • Joerg Standfuss
    • 2
  1. 1.MRC Laboratory of Molecular BiologyCambridgeUK
  2. 2.Laboratory of Biomolecular ResearchPaul Scherrer InstituteVilligenSwitzerland

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