Discovery of Cyclic Peptide Binders from Chemically Constrained Yeast Display Libraries

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Engineering

Background:

• Cyclic peptides engineered for protein binding offer therapeutic and diagnostic potential due to high affinity and selectivity.
• Traditional methods for isolating cyclic peptide binders rely on phage display and panning selections.
• Limitations exist in current methods for efficient identification and characterization of cyclic peptide binders.

Purpose of the Study:

• To establish and detail a yeast surface display platform for identifying and characterizing cyclic peptide binders.
• To provide an alternative to phage display for generating genetically encoded combinatorial libraries of cyclic peptides.
• To demonstrate the utility of yeast display for high-affinity binder isolation and binding affinity estimation.

Main Methods:

• Development of a yeast surface display system for expressing linear peptide precursors.
• In situ cyclization of displayed linear peptides using disuccinimidyl glutarate.
• Isolation of high-affinity cyclic peptide binders using magnetic selection and fluorescence-activated cell sorting (FACS).

Main Results:

• Efficient isolation of high-affinity cyclic peptide binders from yeast display libraries was achieved.
• Protocols for synthesizing yeast display libraries and cyclizing displayed peptides were detailed.
• Yeast surface displayed cyclic peptides enabled efficient binding affinity estimation, reducing reliance on synthesized peptides.

Conclusions:

• The yeast surface display platform provides an effective alternative for discovering and characterizing cyclic peptide binders.
• This method streamlines the process of identifying protein-binding cyclic peptides with potential therapeutic applications.
• The platform facilitates robust binding affinity measurements, crucial for mutant characterization and drug development.