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 only sample regions of sequence space near an initial hit. To overcome these challenges, we built a yeast surface displayed 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.