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.