Analysis of Tat Targeting Function and Twin-Arginine Signal Peptide Activity in Escherichia coli

  • Tracy Palmer
  • Ben C. Berks
  • Frank Sargent
Part of the Methods in Molecular Biology book series (MIMB, volume 619)


The Tat system is a protein export system dedicated to the transport of folded proteins across the prokaryotic cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. Proteins are targeted for export by the Tat system via N-terminal signal peptides harbouring an S-R-R-x-F-L-K ‘twin-arginine’ motif. In this chapter qualitative and quantitative assays for native Tat substrates in the model organism Escherichia coli are described. Genetic screening methods designed to allow the rapid positive selection of Tat signal peptide activity and the first positive selection for mutations that inactivate the Tat pathway are also presented. Finally isothermal titration calorimetry (ITC) methods for measuring the affinity of twin-arginine signal peptide–chaperone interactions are discussed.

Key words

Tat system twin-arginine signal peptide TMAO reductase hydrogenase chaperone protein–protein interaction 



We would particularly like to thank Prof. Gary Sawers (Halle-Wittenberg) for his help with developing some of the early methods for analysis of the E. coli Tat system, and all the members of our laboratories past and present. Work in our laboratories is or has been funded by the BBSRC, the MRC, the Wellcome Trust, the European Union and the Royal Society.


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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Tracy Palmer
    • 1
  • Ben C. Berks
    • 2
  • Frank Sargent
    • 1
  1. 1.Division of Molecular Microbiology, College of Life SciencesUniversity of DundeeDundeeScotland
  2. 2.Department of BiochemistryOxford UniversityOxfordUK

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