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Enzymatic Assembly of Ubiquitin Chains

  • Martin A. Michel
  • David KomanderEmail author
  • Paul R. ElliottEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1844)

Abstract

The availability of different polyubiquitin chains of specific linkage types has changed the appreciation of the specificity in the ubiquitin (Ub) system. Numerous E2 Ub-conjugating enzymes and E3 Ub ligases, Ub-binding domains (UBDs), and deubiquitinases (DUBs) are now known to assemble, bind, or hydrolyze individual linkage types, respectively. Biochemical and structural studies of these processes require milligram quantities of pure polyUb. Here we describe protocols that allow the enzymatic synthesis and purification of six of the eight homotypic polyUb chains through the use of chain-specific Ub ligases and DUBs.

Key words

Ubiquitin Deubiquitinase Ligase Linkage-specific Enzymatic assembly Protein purification 

Notes

Acknowledgments

We would like to thank present and past members of the DK laboratory who have helped to develop and refine described protocols by experimentation and discussions. Work in the D.K. lab is funded by the Medical Research Council [U105192732], the European Research Council [309756, 724804], and the Lister Institute for Preventive Medicine. M.A.M. was supported by a PhD fellowship of the Boehringer Ingelheim Fonds and a Doc.Mobility fellowship of the Swiss National Science Foundation.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Division of Protein and Nucleic Acid ChemistryMRC Laboratory of Molecular BiologyCambridgeUK

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