In-Cell Approach to Evaluate E3 Ligases for Use in Targeted Protein Degradation.

ABSTRACT: A major challenge in evaluating the suitability of ∼700 known and putative E3 ligases for target protein degradation (TPD) is the lack of ligase-specific binders. Here, we use genetic code expansion (GCE) to express in living cells an E3 ligase with a site-specifically encoded, tetrazine-containing noncanonical amino acid (Tet-ncAA). Then, using click chemistry, we conjugate the incorporated Tet with a strained trans-cyclooctene (sTCO) tethered to a neosubstrate protein binder. The resulting covalent E3 ligase-binder construct can then be evaluated for the TPD of the neosubstrate. We first demonstrate that cereblon (CRBN) has a rather high plasticity for TPD by studying CRBN containing Tet-ncAA at a variety of surface positions. When these CRBN forms are covalently tethered to an sT

SUBMITTER: Zheng Y 

PROVIDER: S-EPMC12684378 | biostudies-literature | 2025 Jun

REPOSITORIES: biostudies-literature