Decoding Protein–Peptide Interactions Using a Large, Target-Agnostic Yeast Surface Display Library

Protein-peptide interactions underlie key biological processes and are commonly utilized in biomedical research and therapeutic discovery. It is often desirable to identify peptide sequence properties that confer high-affinity binding to a target protein. However, common approaches to such characterization are typically low throughput and sample only regions of sequence space near an initial hit. To overcome these challenges, we built a yeast surface display library representing ∼6.1 × 10⁹ unique peptides. We then performed screens against diverse protein targets, including two antibodies, an E3 ubiquitin ligase, and an essential membrane-bound bacterial enzyme. In each case, we observed motifs that appear to drive peptide binding, and we identified multiple novel, high-affinity clones. These results highlight the library's utility as a robust and versatile tool for discovering peptide ligands and for characterizing protein-peptide binding interactions more generally. To enable further studies, we will make the library freely available upon request.