Discovery of Cyclic Peptide Binders from Chemically Constrained Yeast Display Libraries

Cyclic peptides with engineered protein-binding activity have great potential as therapeutic and diagnostic reagents owing to their favorable properties, including high affinity and selectivity. Cyclic peptide binders have generally been isolated from phage display combinatorial libraries utilizing panning based selections. As an alternative, we have developed a yeast surface display platform to identify and characterize cyclic peptide binders from genetically encoded combinatorial libraries. Through a combination of magnetic selection and fluorescence-activated cell sorting (FACS), high-affinity cyclic peptide binders can be efficiently isolated from yeast display libraries. In this platform, linear peptide precursors are expressed as yeast surface fusions. To achieve cyclization of the linear precursors, the cells are incubated with disuccinimidyl glutarate, which crosslinks amine groups within the displayed linear peptide sequence. Here, we detail protocols for cyclizing linear peptides expressed as yeast surface fusions. We also discuss how to synthesize a yeast display library of linear peptide precursors. Subsequently, we provide suggestions on how to utilize magnetic selections and FACS to isolate cyclic peptide binders for target proteins of interest from a peptide combinatorial library. Lastly, we detail how yeast surface displayed cyclic peptides can be used to obtain efficient estimates of binding affinity, eliminating the need for chemically synthesized peptides when performing mutant characterization.