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Probing the Activity of Eukaryotic Rhomboid Proteases In Vitro.

Authors
  • Cordier, B1
  • Lemberg, M K2
  • 1 Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Heidelberg, Germany.
  • 2 Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Heidelberg, Germany. Electronic address: [email protected]
Type
Published Article
Journal
Methods in enzymology
Publication Date
2017
Volume
584
Pages
99–126
Identifiers
DOI: 10.1016/bs.mie.2016.09.045
PMID: 28065274
Source
Medline
Keywords
License
Unknown

Abstract

Proteolysis within the membrane is a recent concept in biology. Rhomboid intramembrane serine proteases are conserved in evolution and serve as key switches in diverse cellular pathways ranging from signaling to protein degradation. Since deregulation of intramembrane proteolysis can lead to severe diseases including neurodegenerative disorders, dissecting their enzymatic function and specificity becomes crucial. As membrane proteins, their solubilization, and purification are technically challenging. As a start point for a comprehensive in vitro characterization of eukaryotic rhomboid proteases, we depict in this chapter a robust workflow to find the best conditions to obtain pure and active enzymes from a bacterial expression system. To monitor the integrity of their active site and visualize substrate cleavage, various established activity assays including activity-based labeling and gel-based cleavage assays are described. These methods are illustrated by use of the Escherichia coli rhomboid protease GlpG and human RHBDL2 as an example.

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