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.