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

中文翻译：

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通过全质粒PCR产物的自连接产生大肽噬菌体展示文库

用于开发基于肽和蛋白质的靶标特异性结合物的噬菌体展示的成功取决于所筛选文库的大小和多样性，但生成大型噬菌体编码多肽库仍然具有挑战性。近年来开发的新肽噬菌体展示文库很少包含超过10亿个克隆，这似乎已成为通过合理努力生成的文库的大小上限。在这里，我们建立了基于全质粒PCR和自连接的策略来克隆2×10 ¹⁰以上的文库成员。通过PCR扩增整个载体DNA（省去了从细菌细胞中分离载体的步骤），以及通过PCR引物附加编码肽库的DNA的DNA，可以实现巨大的文库大小，从而可以通过末端连接实现有效的DNA环化以促进DNA片段载体插入的困难步骤。针对目标物淘选肽库产生了高亲和力的配体，并验证了文库​​的质量，从而验证了新的文库克隆策略。这种简单而有效的策略为非专业研究人员提供了更大的库，从而有望扩展噬菌体展示应用的可能目标。