Purification of E1 and E1-Like Enzymes

Ubiquitin-activating enzyme is the archetype for a family of enzymes catalyzing the ATP-coupled activation of ubiquitin and other class 1 ubiquitin-like proteins required for their subsequent conjugation to cellular targets. The general physical and mechanistic features of the E1 family appear well conserved. Formation of an obligatory E1-ubiquitin thiol ester intermediate forms the basis of a one-step covalent purification of the enzyme on ubiquitin-linked affinity columns that has been adapted for the isolation of E1 paralogs. We describe the facile purification of active E1 from outdated human red blood cells in yields (2–4 nmol/U of blood) that make this an attractive alternative to expression of the proteolytically labile recombinant protein. In addition, two stoichiometric activity assays are described that rely on formation of the E1 ¹²⁵ I-ubiquitin thiol ester and ubiquitin [2,8-³ H]adenylate intermediates.