Synthesis and purification of linkage-specific polyubiquitin chains of distinct length for structural studies

Polyubiquitylation is one of the most versatile post-translational modifications involved in the regulation of numerous intracellular signaling processes. An assembly procedure that is simple, robust, and efficient to synthesize and purify linkage-specific polyubiquitin chains of defined length at a preparative scale is required in biophysical and structural studies. Here, we have optimized known enzymatic procedures in the form of a protocol to obtain multi-milligrams of Lys48-and Lys63-linked polyubiquitin chain types with more than 99% purity. Mass spectrometry (ESI/MS) analysis of K48- and K63-linked diubiquitin confirmed that the enzymes used in the preparation generated homogeneous linkages with no promiscuity.