Genetic Code Expansion Facilitates Programmable Ubiquitylation via UBE2W

Deciphering the ubiquitin code requires homogenous, site-specifically ubiquitylated proteins, yet their access remains difficult. Current chemical and enzymatic methods are often limited by harsh conditions, low yields, or the introduction of non-native scars. Here, we present UbyW (Ubiquitylation by UBE2W), a chemoenzymatic platform that overcomes these hurdles. By repurposing the E2 enzyme UBE2W to target a genetically encoded non-canonical amino acid, a near-native Ub-protein conjugate is formed with high efficiency and site-specificity. The method is broadly applicable to diverse proteins, including those with structured domains, and can be performed in vitro or within a reconstituted cascade in living E. coli for streamlined production. Crucially, UbyW allows for the direct installation of bioorthogonal and photocrosslinking handles at the linkage site, providing powerful tools to probe the functional consequences of ubiquitylation and map ubiquitin-dependent interactomes.