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This protocol details the use of yeast surface display technology for the in vitro-directed evolution of disulfide-tethered macrocyclic peptide ligands. In the first section, we describe the generation of large naïve combinatorial libraries encoding cysteine-rich peptide sequences expressed on the yeast cell surface using homologous recombination-based methods. In the second section, we detail the use of fluorescence-activated cell sorting to rapidly and effectively isolate yeast-encoding disulfide-tethered macrocyclic peptide ligands with favorable binding properties. In the last section, we describe the quantitative characterization of isolated disulfide-tethered macrocyclic peptide ligand variants directly as yeast cell surface fusions, thus eliminating the need for costly and time-consuming synthesis and purification.

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Quantitative Affinity Screening of Macrocyclic Peptide Libraries Using Yeast Surface Display Technology

  • 实验方案

  • 发表日期：

    2025年

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  • Ylenia Mazzocato,
  • Zhanna Romanyuk,
  • Monica Chinellato,
  • Camilla Mazzucco,
  • Linda Trevisan,
  • Sara Linciano,
  • Alessandro Angelini

摘要

This protocol details the use of yeast surface display technology for the in vitro-directed evolution of disulfide-tethered macrocyclic peptide ligands. In the first section, we describe the generation of large naïve combinatorial libraries encoding cysteine-rich peptide sequences expressed on the yeast cell surface using homologous recombination-based methods. In the second section, we detail the use of fluorescence-activated cell sorting to rapidly and effectively isolate yeast-encoding disulfide-tethered macrocyclic peptide ligands with favorable binding properties. In the last section, we describe the quantitative characterization of isolated disulfide-tethered macrocyclic peptide ligand variants directly as yeast cell surface fusions, thus eliminating the need for costly and time-consuming synthesis and purification.