Ubiquitin Chain Synthesis
Several important signaling processes depend on the tagging of cellular proteins with “polyubiquitin chains”—ubiquitin polymers whose building blocks are connected by isopeptide bonds between G76 of one ubiquitin and a specific lysine residue of the next one. Here we describe procedures for the synthesis of polyubiquitin chains of defined lengths that are linked through the K48 or K63 side chains. The method involves a series of enzymatic reactions in which proximally and distally blocked monoubiquitins (or chains) are conjugated to produce a particular chain in high yield. Individual chains are then deblocked and joined in another round of reaction. Successive rounds of deblocking and synthesis can give rise to a chain of any desired length.
Key WordsConjugating enzyme conjugation deubiquitinating enzymes isopeptide polyubiquitin (chain) ubiquitin
We thank R. Cohen for providing the YUH1 expression clone and for a critical reading of the manuscript.
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