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In Vitro and In Vivo Approaches to Studying the Bacterial Signal Peptide Processing

  • Peng Wang
  • Ross E. Dalbey
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 619)

Abstract

Protein targeting in both eukaryotic and prokaryotic cells is often directed by a signal sequence located at the amino-terminus of the protein. In eukaryotes, proteins that are sorted into different compartments of the cell, such as endoplasmic reticulum, mitochondria, and chloroplast, require different signal sequences. In bacteria, proteins which are exported to the outer membrane or the periplasmic space are also guided by signal peptides. After the protein is translocated across the cytoplasmic membrane, the signal peptide is proteolytically removed by signal peptide cleavage. Here, in this chapter, we describe methods to study signal peptide processing in bacteria, including purification of signal peptidase and its substrates. We also describe the measurement of the catalytic constants of signal peptidases using an in vitro assay. In addition, we will present an in vivo assay using a temperature sensitive signal peptidase strain to determine which preproteins are processed by Signal peptidase 1.

Key words

Signal peptide signal peptidase purification activity assay preprotein 

Notes

Acknowledgment

The work was supported by National Institute of Health Grant (GM63862-05) to R.E.D

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Peng Wang
    • 1
  • Ross E. Dalbey
    • 1
  1. 1.Department of ChemistryThe Ohio State UniversityColumbusUSA

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