Phage-Assisted Continuous Selection of Bioactive Cyclic Peptides.

Cyclic peptides (CPs) are a class of molecules that can display various biological activities and have been utilized extensively as a source for therapeutic leads and chemical probes. Recently, a strategy inspired by phage-assisted continuous evolution (PACE) was established for high-throughput discovery of CP protein aggregation inhibitors. This method leverages customizable genetic circuits to link desired activities of CP to expression of M13 phage minor coat protein pIII, allowing enrichment of active CP sequences by serial phage propagations through E. coli host cells. Compared with existing strategies, this approach enables rapid cellular selection of bioactive CPs while requiring only standard molecular biology techniques and equipment. In this chapter, we provide a comprehensive protocol for applying this strategy to select CP inhibitors against aggregation-prone proteins of interest. Details and procedures covered in this protocol can also provide a foundation for experimental design to discover CPs possessing other bioactivities.