Gu Yu, Iannuzzelli Jacob A, Fasan Rudi
Department of Chemistry, University of Rochester, Rochester, NY, USA.
Methods Mol Biol. 2022;2371:261-286. doi: 10.1007/978-1-0716-1689-5_14.
Macrocyclic peptides represent promising scaffolds for targeting biomolecules with high affinity and selectivity, making methods for the diversification and functional selection of these macrocycles highly valuable for drug discovery purposes. We recently reported a novel phage display platform (called MOrPH-PhD) for the creation and functional exploration of combinatorial libraries of genetically encoded cyclic peptides. In this system, spontaneous, posttranslational peptide cyclization by means of a cysteine-reactive non-canonical amino acid is integrated with M13 bacteriophage display, enabling the creation of genetically encoded macrocyclic peptide libraries displayed on phage particles. Using this system, it is possible to rapidly generate and screen large libraries of phage-displayed macrocyclic peptides (up to 10 to 10 members) in order to identify high-affinity binders of a target protein of interest. Herein, we describe step-by-step protocols for the production of MOrPH-PhD libraries, the screening of these libraries against an immobilized protein target, and the isolation and characterization of functional macrocyclic peptides from these genetically encoded libraries.
大环肽是用于以高亲和力和选择性靶向生物分子的有前景的支架，因此这些大环化合物的多样化和功能选择方法对于药物发现目的具有很高的价值。我们最近报道了一种新型噬菌体展示平台（称为MOrPH-PhD），用于创建和功能探索遗传编码环肽的组合文库。在该系统中，通过半胱氨酸反应性非天然氨基酸进行的自发翻译后肽环化与M13噬菌体展示相结合，能够创建展示在噬菌体颗粒上的遗传编码大环肽文库。使用该系统，可以快速生成和筛选噬菌体展示大环肽的大型文库（多达10至10个成员），以鉴定感兴趣的靶蛋白的高亲和力结合剂。在此，我们描述了生产MOrPH-PhD文库、针对固定化蛋白靶标筛选这些文库以及从这些遗传编码文库中分离和表征功能性大环肽的逐步方案。
