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Updated: Jun 2, 2025
Protein Engineering by Yeast Surface Display
Published on: November 29, 2024
Directed Evolution of Multicyclic Peptides Using Yeast Display for Sensitive and Selective Fluorescent Analysis of
Chaoying Xu1,2, Xiaoting Meng1,3, Ping Chai1
1The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China.
Abstract:
CD28 is a costimulatory receptor that provides the second signal necessary for T-cell activation and is associated with diseases, including rheumatoid arthritis, asthma, and cancer. Targeting CD28 is crucial for both functional bioanalysis and therapeutic development. Molecular probes, particularly fluorescent probes, can enhance our understanding of CD28's cellular roles. However, existing antibody-based probes face challenges such as high production costs, low stability, and large size, which limit their bioanalytical applications. Thus, there is a need for smaller, robust probes that enable the sensitive and selective targeting of CD28. Multicyclic peptides have emerged as promising candidates for novel therapeutics and molecular probes. Recently, we identified disulfide-directed multicyclic peptides (DDMPs) that bind CD28 with submicromolar affinity; however, their relatively low affinity limits further applications. In this study, we develop a DDMP evolving system based on yeast display and error-prone PCR to identify high-affinity peptide binders. We obtained DDMPs with a picomolar affinity for CD28, exceptional binding specificity, and remarkable oxidative folding efficiency. Furthermore, we developed fluorescent probes and labeling strategies for detecting and visualizing CD28 expression in human T cells. This advancement opens new avenues for studying T-cell dynamics and activation states, which are essential for understanding immune responses and developing targeted therapies. Our study not only produces potent CD28 binders and probes but also establishes a robust platform for optimizing other multicyclic peptide-based probes and therapeutics.
Insights
Researchers developed high-affinity peptide probes targeting CD28, a key molecule in T-cell activation. These smaller, stable probes improve T-cell analysis and therapeutic development for immune-related diseases.
Area of Science:
- Immunology
- Biotechnology
- Molecular Biology
Background:
- CD28 is a crucial costimulatory receptor for T-cell activation, implicated in diseases like rheumatoid arthritis, asthma, and cancer.
- Current antibody-based probes for CD28 face limitations including high cost, poor stability, and large size, hindering bioanalytical applications.
- There is a need for smaller, robust, and highly specific probes for sensitive CD28 detection and targeting.
Purpose of the Study:
- To develop a novel system for evolving high-affinity disulfide-directed multicyclic peptides (DDMPs) targeting CD28.
- To create advanced molecular probes for sensitive and selective detection and visualization of CD28 expression in human T cells.
- To establish a robust platform for optimizing peptide-based probes and therapeutics.
Main Methods:
- Utilized a yeast display and error-prone PCR system to evolve DDMPs for CD28 binding.
- Characterized the affinity, specificity, and oxidative folding efficiency of the evolved DDMPs.
- Developed fluorescent probes and labeling strategies for CD28 detection in human T cells.
Main Results:
- Identified DDMPs with picomolar affinity and exceptional binding specificity for CD28.
- Achieved remarkable oxidative folding efficiency in the developed peptide probes.
- Successfully developed fluorescent probes for visualizing CD28 expression in human T cells.
Conclusions:
- The developed DDMPs offer potent and specific targeting of CD28, overcoming limitations of existing probes.
- The novel probes enable sensitive detection and visualization of CD28, advancing T-cell dynamics studies.
- This work establishes a versatile platform for optimizing peptide-based probes and therapeutics for immune-related applications.
