Enzymatic Assembly of Ubiquitin Chains
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 wordsUbiquitin Deubiquitinase Ligase Linkage-specific Enzymatic assembly Protein purification
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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