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Site-Specific Cross-Linking of In Vitro Synthesized E. coli Preproteins for Investigating Transmembrane Translocation Pathways

  • Sascha Panahandeh
  • Matthias Müller
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 619)

Abstract

A method is described for the preparation and usage of an E. coli cell-free translation system primed to incorporate the commercially available photoreactive analogue of phenyalanine, pBpa, into newly synthesized proteins. Incorporation is achieved by means of an amber suppressor tRNA specifically charged with pBpa. The method is exemplified for the site-specific photocross-linking of the signal sequence of a Tat (twin-arginine translocation) precursor protein to the Tat translocase in the cytoplasmic membrane of E. coli.

Key words

Site-specific cross-linking photoprobes p-benzoyl-phenylalanine amber suppressor twin-arginine translocation Tat protein export in vitro transcription-translation system inner membrane vesicles Escherichia coli 

Notes

Acknowledgments

We gratefully acknowledge Dr. Peter Schultz, The Scripps Research Institute, La Jolla, for providing suppressor plasmids. This work was supported by grant LSHG-CT-2004-05257 of the European Union and grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 388 and Graduiertenkolleg 434).

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Sascha Panahandeh
    • 1
  • Matthias Müller
    • 2
  1. 1.Institute of Biochemistry and Molecular Biology, ZBMZUniversity of FreiburgFreiburgGermany
  2. 2.Institute of Biochemistry and Molecular Biology, ZBMZUniversity of FreiburgFreiburgGermany

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