Generation of a 100-billion cyclic peptide phage display library having a high skeletal diversity

Phage display is a powerful technique routinely used for the generation of peptide- or protein-based ligands. The success of phage display selections critically depends on the size and structural diversity of the libraries, but the generation of large libraries remains challenging. In this work, we have succeeded in developing a phage display library comprising around 100 billion different (bi)cyclic peptides and thus more structures than any previously reported cyclic peptide phage display library. Building such a high diversity was achieved by combining a recently reported library cloning technique, based on whole plasmid PCR, with a small plasmid that facilitated bacterial transformation. The library cloned is based on 273 different peptide backbones and thus has a large skeletal diversity. Panning of the peptide repertoire against the important thrombosis target coagulation factor XI enriched high-affinity peptides with long consensus sequences that can only be found if the library diversity is large.