Expression, purification, and properties of the Ubc4/5 family of E2 enzymes.
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.
Lorick
Kevin L
KL
Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
Jensen
Jane P
JP
Weissman
Allan M
AM
D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C489270:UBC5 protein, S cerevisiae; C501306:Ubc4 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes