Probing the activity of eukaryotic rhomboid proteases in vitro
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...
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| Main Authors: | , |
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| Format: | Chapter/Article |
| Language: | English |
| Published: |
2017
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| In: |
Enzymology at the membrane interface - intramembrane proteases
Year: 2016, Pages: 99-126 |
| DOI: | 10.1016/bs.mie.2016.09.045 |
| Online Access: | Verlag, Volltext: https://doi.org/10.1016/bs.mie.2016.09.045 Verlag, Volltext: http://www.sciencedirect.com/science/article/pii/S0076687916303184 
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| Author Notes: | B. Cordier, M.K. Lemberg |
| Summary: | 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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| Item Description: | Available online 27 October 2016 Gesehen am 14.09.2018 |
| Physical Description: | Online Resource |
| ISBN: | 9780128122143 0128122145 9780128122136 |
| DOI: | 10.1016/bs.mie.2016.09.045 |