Ubiquitin is a 76 amino acid (aa) protein that is ubiquitously expressed in all eukaryotic organisms. Ubiquitin is highly conserved with 96% aa sequence identity shared between human and yeast Ubiquitin, and 100% aa sequence identity shared between human and mouse Ubiquitin (1). In mammals, four Ubiquitin genes encode for two Ubiquitin-ribosomal fusion proteins and two poly-Ubiquitin proteins. Cleavage of the Ubiquitin precursors by deubiquitinating enzymes gives rise to identical Ubiquitin monomers each with a predicted molecular weight of 8.6 kDa. Conjugation of Ubiquitin to target proteins involves the formation of an isopeptide bond between the C-terminal glycine residue of Ubiquitin and a lysine residue in the target protein. This process of conjugation, referred to as ubiquitination or ubiquitylation, is a multi-step process that requires three enzymes: a Ubiquitin-activating (E1) enzyme, a Ubiquitin-conjugating (E2) enzyme, and a Ubiquitin ligase (E3). Ubiquitination is classically recognized as a mechanism to target proteins for degradation and as a result, Ubiquitin was originally named ATP-dependent Proteolysis Factor 1 (APF-1) (2,3). In addition to protein degradation, ubiquitination has been shown to mediate a variety of biological processes such as signal transduction, endocytosis, and post-endocytic sorting (4-7).
Mature forms of Ubiquitin have a highly conserved diglycine motif at the carboxyl terminus which is crucial for activity and recognition in conjugation and deconjugation reactions. The replacement of this diglycine peptide with two alanine residues results in an inactive Ubiquitin. This Ubiquitin cannot be activated by the Ubiquitin-activating (E1) enzyme in an ATP-dependent manner, is not capable of subsequent thioester interaction with Ubiquitin-conjugating (E2) enzymes and/or Ubiquitin ligases (E3), and is thus not capable of forming isopeptide bonds or Ubiquitin conjugates. It can be used as a negative control in conjugation reactions, or in binding studies with Ubiquitin-activating (E1) enzymes, Ubiquitin-conjugating (E2) enzymes, Ubiquitin Ligases (E3), and DUB enzymes or other proteins that interact with Ubiquitin via Ubiquitin associated domains (UBAs) or Ubiquitin-interacting motifs (UIMs). This protein contains an N-terminal His6-tag.
