Avidity-Based Method for the Efficient Generation of Monoubiquitinated Recombinant Proteins

Spencer L. Nelson, Yunan Li, Yue Chen, Lalit Deshmukh

Research output: Contribution to journalArticlepeer-review

5 Scopus citations


Monoubiquitination of proteins governs diverse physiological processes, and its dysregulation is implicated in multiple pathologies. The difficulty of preparing sufficient material often complicates the biophysical studies of monoubiquitinated recombinant proteins. Here we describe a robust avidity-based method that overcomes this problem. As a proof-of-concept, we produced milligram quantities of two monoubiquitinated targets, Parkinson’s protein α-synuclein and ESCRT-protein ALIX, using NEDD4-family E3 ligases. Monoubiquitination hotspots were identified by quantitative chemical proteomics. Using FRAP and dye-binding assays, we uncovered strikingly opposite effects of monoubiquitination on the phase separation and fibrillization properties of these two amyloidogenic proteins, reflecting differences in their intermolecular interactions, thereby providing unique insights into the impact of monoubiquitination on protein aggregation.

Original languageEnglish (US)
Pages (from-to)7748-7752
Number of pages5
JournalJournal of the American Chemical Society
Issue number14
StatePublished - Apr 12 2023

Bibliographical note

Publisher Copyright:
© 2023 The Authors. Published by American Chemical Society.

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.


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