Preparation and purification of mono-ubiquitinated proteins using Avi-tagged ubiquitin

We describe the purification of natively mono-ubiquitinated FANCI:FANCD2 complex, along with other mono-ubiquitinated components of the Fanconi anemia -BRCA pathway

Winnie Tan


Scholarcy highlights

  • Ubiquitination is the process of covalent attachment of ubiquitin to a substrate protein
  • In order to purify natively mono-ubiquitinated proteins, we engineered a bacterial expression construct that encodes human ubiquitin with: 1) an N-terminal 10xHis tag for affinity purification, 2) an Avi-tag for site-specific biotinylation and biotin-affinity purification, 3) a 3C protease site for site-specific protease cleavage, leaving native ubiquitin with one artefactual Nterminal proline,z and 4) a second ORF for co-expression of BirA biotin ligase for efficient bacterial biotinylation of recombinant protein
  • The single-step nature of affinity purification followed by protease-mediated elution of the modified proteins make this technique amenable for use in the study of any ubiquitinated protein of research interest
  • We focused on the Fanconi anemia-BRCA pathway and were able to purify mono-ubiquitinated FANCI: FANCD2 complex, PCNA trimer and nucleosomes which can be used as an reagent to answer long-standing questions in the field
  • Previous protocols for purifying ubiquitinated proteins involve engineering a tandem ubiquitin-binding domain of interest or immunoprecipitation of ubiquitinated peptides using anti-ubiquitin, these methods often contain a mixture of poly- and mono-ubiquitination proteins
  • Enrichment of ubiquitinated peptides and mass spectrometry-based approach are required for protein identification, hindering identification of low abundance ubiquitinated protein complexes
  • Preparation and purification of mono-ubiquitinated proteins using Avi-tagged ubiquitin spectrum represent mainly single-event preferential cleavage of the peptide bonds resulting in the sequence information recorded simultaneously from both the N- and C-termini of the peptide

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