Generation of a Large Peptide Phage Display Library\nby Self-Ligation of Whole-Plasmid PCR Product

The\nsuccess of phage display, used for developing target-specific\nbinders based on peptides and proteins, depends on the size and diversity\nof the library screened, but generating large libraries of phage-encoded\npolypeptides remains challenging. New peptide phage display libraries\ndeveloped in recent years rarely contained more than 1 billion clones,\nwhich appears to have become the upper size limit for libraries generated\nwith reasonable effort. Here, we established a strategy based on whole-plasmid\nPCR and self-ligation to clone a library with more than 2 × 10¹⁰ members. The enormous library size could be obtained through\namplifying the entire vector DNA by PCR, which omitted the step of\nvector isolation from bacterial cells, and through appending DNA coding\nfor the peptide library via a PCR primer, which enabled efficient\nDNA circularization by end-ligation to facilitate the difficult step\nof vector-insertion of DNA fragments. Panning the peptide repertoires\nagainst a target yielded high-affinity ligands and validated the quality\nof the library and thus the new library cloning strategy. This simple\nand efficient strategy places larger libraries within reach for nonspecialist\nresearchers to hopefully expand the possible targets of phage display\napplications.