UBE2W (Ubc16) [6His-tagged]

Ubiquigent

Catalog No: UBI-62-0085-020

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Species Reactivity: Human  
Source: E. coli expression  
Formula: 50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol  
Concentration: 1 mg/ml  
Molecular Weight: ~22.2 kDa  
Protein Sequence: Accession number: NP_001001481.1. For full protein sequence information download the Certificate of Analysis pdf.  
Quality Control Protein Identification: Confirmed by mass spectrometry.  
Quality Control Activity: E2-Ubiquitin Thioester Loading Assay: The activity of His-UBE2W was validated by loading E1 UBE1 activated ubiquitin onto the active cysteine of the His-UBE2W E2 enzyme via a transthiolation reaction. Incubation of the UBE1 and His-UBE2W enzymes in the presence of ubiquitin and ATP at 30 °C was compared at two time points, T0 and T10 minutes. Sensitivity of the ubiquitin/His-UBE2W thioester bond to the reducing agent DTT was confirmed.  

Background 

The enzymes of the ubiquitylation pathway play a pivotal role in a number of cellular processes including the regulated and targeted proteasomal degradation of substrate proteins. Three classes of enzymes are involved in the process of ubiquitylation; activating enzymes (E1s), conjugating enzymes (E2s) and protein ligases (E3s). UBE2W is a member of the E2 conjugating enzyme family and cloning of the human gene was first described by Yin et al. (2006). UBE2W comprises 7 exons and there are two Nuclear Localisation Signals (NLS) located in the c-terminus of the UBC domain (Yin et al., 2006). Interaction of UBE2W with the human heteromeric RING E3 BRCA1-BARD1 has been demonstrated using yeast two-hybrid screening. UBE2W binds directly to the RING motif of BRCA-1 causing autoubiquitylation of BRCA1-BARD1 and monoubiquitylation of BRCA1 alone in vitro (Christensen et al., 2007). UBE2W also interacts with UBE1 and the E3 ligase FANCL to monoubiquitylate FANCD2 in vitro (Alpi et al., 2008)

References:

Alpi AF, Pace PE, Babu MM, Patel KJ (2008) Mechanistic insight into site-restricted monoubiquitination of FANCD2 by Ube2t, FANCL, and FANCI. Mol Cell 32, 767-77.
Christensen DE, Brzovic PS, Klevit RE (2007) E2-BRCA1 RING interactions dictate synthesis of mono- or specific polyubiquitin chain linkages. Nat Struct Mol Biol 14, 941-8.
Yin G, Ji C, Wu T, Shen Z, Xu X, Xie Y, Mao Y (2006) Cloning, characterization and subcellular localization of a gene encoding a human Ubiquitin-conjugating enzyme (E2) homologous to the Arabidopsis thaliana UBC-16 gene product. Front Biosci 11, 1500-7.