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Updated: Aug 15, 2026

In Vitro Analysis of E3 Ubiquitin Ligase Function
Published on: May 14, 2021
Expression, purification, and properties of the Ubc4/5 family of E2 enzymes
Kevin L Lorick1, Jane P Jensen, Allan M Weissman
1Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
Abstract:
Ubiquitin-conjugating enzymes (E2s) play a central role in ubiquitylation. They function to bridge the first, nonspecific step of ubiquitin activation by E1 with the transfer of activated ubiquitin to substrates by substrate-specific E3s. While sharing a common core UBC domain, members of this family exhibit significant specificity in their physical and functional interactions with E3s. Among the families of E2s, members of the yeast Ubc4/5 family are particularly well conserved in higher metazoans. In humans, these are represented by the UbcH5 family. Members of this ubiquitously expressed family show a capacity to interact with a wide range of E3s from both HECT and RING finger families, making them particularly useful tools in the laboratory. Using the UbcH5 family as a prototype, this chapter describes methods for the expression, purification, and characterization of E2 enzymes in vitro and some of the basics for their use in experiments in cells.
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