Controlled Synthesis of Polyubiquitin Chains

Many intracellular signaling processes depend on the modification of proteins with polymers of the conserved 76‐residue protein ubiquitin. The ubiquitin units in such polyubiquitin chains are connected by isopeptide bonds between a specific lysine residue of one ubiquitin and the carboxyl group of G76 of the next ubiquitin. Chains linked through K48‐G76 and K63‐G76 bonds are the best characterized, signaling proteasome degradation and nonproteolytic outcomes, respectively. The molecular determinants of polyubiquitin chain recognition are under active investigation; both the chemical structure and the length of the chain can influence signaling outcomes. In this article, we describe the protein reagents necessary to produce K48‐ and K63‐linked polyubiquitin chains and the use of these materials to produce milligram quantities of specific‐length chains for biochemical and biophysical studies. The method involves reactions catalyzed by linkage‐specific conjugating factors, in which proximally and distally blocked monoubiquitins (or chains) are joined to produce a particular chain product in high yield. Individual chains are then deblocked and joined in another round of reaction. Successive rounds of deblocking and synthesis give rise to a chain of the desired length.