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Purification and Functional Reconstitution of the Bacterial Protein Translocation Pore, the SecYEG Complex

  • Ilja Kusters
  • Geert van den Bogaart
  • Janny de Wit
  • Viktor Krasnikov
  • Bert Poolman
  • Arnold Driessen
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 619)

Abstract

In bacteria, proteins are secreted across the cytoplasmic membrane by a protein complex termed translocase. The ability to study the activity of the translocase in vitro using purified proteins has been instrumental for our understanding of the mechanisms underlying this process. Here, we describe the protocols for the purification and reconstitution of the SecYEG complex in an active state into liposomes. In addition, fluorescence based in vitro assays are described that allow monitoring translocation activity discontinuously and in real time.

Key words

Protein secretion reconstitution SecYEG SecA in vitro translocation fluorescence quenching 

Notes

Acknowledgements

This work was supported by NanoNed, a national nanotechno-logy program coordinated by the Dutch Ministry of Economical Affairs and the Zernike Institute for Advanced Materials.

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Ilja Kusters
    • 1
  • Geert van den Bogaart
    • 2
  • Janny de Wit
    • 1
  • Viktor Krasnikov
    • 3
  • Bert Poolman
    • 2
  • Arnold Driessen
    • 1
  1. 1.Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute and Zernike Institute for Advanced MaterialsUniversity of GroningenGroningenThe Netherlands
  2. 2.Department of Membrane Enzymology, Groningen Biomolecular Sciences and Biotechnology Institute and Zernike Institute for Advanced MaterialsUniversity of GroningenGroningenThe Netherlands
  3. 3.Department of Optical Condensed Matter Physics, Zernike Institute for Advanced MaterialsUniversity of GroningenGroningenThe Netherlands

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