Enabling Protein Degradation Drug Discovery

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  • Name
    Catalogue Number
    Size
    Price
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  • Name:
    UBE2W (Ubc16) [GST-tagged]
    Catalogue Number:
    62-0091-100
    Size:
    100 µg
    Price:
    £325
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  • Species
    Human
  • Source
    E. coli expression
  • Quantity
    100 μg
  • Storage
    -70°C
  • Concentration
    1 mg/ml
  • Formulation
    50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol
  • Molecular Weight
    ~45 kDa
  • Stability
    12 months at -70°C; aliquot as required
  • Protein Sequence
    Accession number: NP_001001481.1. For full protein sequence information download the Certificate of Analysis pdf.
  • QA; Protein Identification
    Confirmed by mass spectrometry.
  • QA; Activity
    E2-Ubiquitin Thioester Loading Assay: The activity of GST-UBE2W was validated by loading E1 UBE1 activated ubiquitin onto the active cysteine of the GST-UBE2W E2 enzyme via a transthiolation reaction. Incubation of the UBE1 and GST-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/GST-UBE2W thioester bond to the reducing agent DTT was confirmed.  

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.