ArticleNature protocols2015
Click chemistry for targeted protein ubiquitylation and ubiquitin chain formation.
Article in Nature protocols, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 35 papers.
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Who cites it
35 citing papers in PubMed.
- Fluorophore placement and size impact E2 ubiquitination efficiency.The Biochemical journal · 2026Article
- Optimization of the Chemical Monoubiquitination System for Low-Solubility Protein: Achieving Balance Between Specificity and Yield.Current issues in molecular biology · 2026Article
- Single-molecule analysis reveals the mechanism of chromatin ubiquitylation by variant PRC1 complexes.Science advances · 2025Article
- Click Chemistry Methodology: The Novel Paintbrush of Drug Design.ACS chemical biology · 2025Review
- A Site-Specific Click Chemistry Approach to Di-Ubiquitylate H1 Variants Reveals Position-Dependent Stimulation of the DNA Repair Protein RNF168.Angewandte Chemie (International ed. in English) · 2024Article
- Genetic Code Expansion Approaches to Decipher the Ubiquitin Code.Chemical reviews · 2024Review
- Interactome of intact chromatosome variants with site-specifically ubiquitylated and acetylated linker histone H1.2.Nucleic acids research · 2024Article
- Functional analysis of protein post-translational modifications using genetic codon expansion.Protein science : a publication of the Protein Society · 2023Review
- Semisynthetic Approach to the Analysis of Tumor Suppressor PTEN Ubiquitination.Journal of the American Chemical Society · 2023Article
- Getting to the Root of Branched Ubiquitin Chains: A Review of Current Methods and Functions.Methods in molecular biology (Clifton, N.J.) · 2023Review
- Generation and Characterization of Site-Specifically Mono-Ubiquitylated p53.Chembiochem : a European journal of chemical biology · 2022Article
- Emerging Roles of Non-proteolytic Ubiquitination in Tumorigenesis.Frontiers in cell and developmental biology · 2022Review
- Deciphering the Structure and Formation of Amyloids in Neurodegenerative Diseases With Chemical Biology Tools.Frontiers in chemistry · 2022Review
- Site-specific ubiquitylation acts as a regulator of linker histone H1.Nature communications · 2021Article
- Site-specific ubiquitination of the E3 ligase HOIP regulates apoptosis and immune signaling.The EMBO journal · 2020Article
- Deciphering protein post-translational modifications using chemical biology tools.Nature reviews. Chemistry · 2020Review
- Branched Ubiquitination: Detection Methods, Biological Functions and Chemical Synthesis.Molecules (Basel, Switzerland) · 2020Review
- The Length of a Ubiquitin Chain: A General Factor for Selective Recognition by Ubiquitin-Binding Proteins.Angewandte Chemie (International ed. in English) · 2020Article
- Protein Engineering in the Ubiquitin System: Tools for Discovery and Beyond.Pharmacological reviews · 2020Review
- Conformational and functional characterization of artificially conjugated non-canonical ubiquitin dimers.Scientific reports · 2019Article
Corrections and comments
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Authors and funding
5 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Herein we describe a simple protocol for the efficient generation of site-specific ubiquitin-protein conjugates using click chemistry. By using two different methods to expand the genetic code, the two bio-orthogonal functionalities that are necessary for Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), an alkyne and an azide, are co-translationally incorporated into the proteins of interest with unnatural amino acids. Protein ubiquitylation is subsequently carried out with the purified proteins in vitro by CuAAC. In addition, we provide a protocol for the incorporation of two unnatural amino acids into a single ubiquitin, resulting in a 'bifunctional' protein that contains both an alkyne and an azide functionality, thereby enabling assembly of free ubiquitin chains as well as ubiquitin chains conjugated to a target protein. Our procedure enables the synthesis of nonhydrolyzable ubiquitin-protein conjugates within 1 week (given that the relevant cDNAs are at hand), and it yields conjugates in milligram quantities from 1-liter expression cultures. The approach described herein is faster and less laborious than other methods, and it requires only standard molecular biology equipment. Moreover, the protocol can be readily adapted to achieve conjugation at any site of any target protein, which facilitates the generation of custom-tailored ubiquitin-protein conjugates.
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Registered trials
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