Controlled synthesis of polyubiquitin chains

Methods Enzymol. 2005;399:21-36. doi: 10.1016/S0076-6879(05)99002-2.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Electrophoresis, Polyacrylamide Gel
  • Polyubiquitin / chemical synthesis*
  • Polyubiquitin / chemistry
  • Polyubiquitin / isolation & purification

Substances

  • Polyubiquitin