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In Vivo Analysis of Protein Translocation to the Escherichia coli Periplasm

  • Dominique Belin
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 619)

Abstract

General protein export requires the cooperation of two elements, the Sec translocase and a signal sequence. The interactions of both wild type and mutant components can be studied in vivo using a number of genetic systems. Signal sequence mutations that prevent export have been characterized (“down mutations”). Suppressors of these signal sequence mutations, known as prl mutations, have been isolated in most sec genes. More recently, inactive N-terminal regions of cytoplasmic proteins were converted into active signal sequences (“up mutations”).

Alkaline phosphatase (PhoA), an enzyme only active after export to the periplasm, provides the best and most versatile quantitative reporter for protein translocation studies. Cleavable signal sequences can be used to monitor protein export in a time frame of 15–120 s. Chimeric proteins expressed from an inducible promoter can be used to measure kinetics of enzyme accumulation in a time frame of 10–100 min. Finally, the export activity of PhoA-chimeras can be visualized in a semi-quantitative way by staining colonies growing on Petri dishes with a chromogenic substrate, in the time frame of 10–40 h.

Key words

Signal sequence alkaline phosphatase E. coli translocase prl mutations gene fusions leader peptide leader peptidase 

Notes

Acknowledgments

The author thanks Luzma Guzman, Alan Derman, and the other members of the Beckwith lab for sharing their enthusiasm and expertise on alkaline phosphatase. Sandrine Bost identified the power of the kinetic assay to study mutant phenotypes. The experiments in the lab were mostly performed with the invaluable help of Filo Silva. This work was supported by grants of the FNS and by the Canton de Genève.

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Dominique Belin
    • 1
  1. 1.Department of Pathology and ImmunologyUniversity Medical Center, University of GenevaGenevaSwitzerland

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