Dombrowsky Carolin S, Hofmann Sarah, Kolmar Harald
Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, Germany.
Centre for Synthetic Biology, Technical University of Darmstadt, Darmstadt, Germany.
Methods Mol Biol. 2025;2934:275-291. doi: 10.1007/978-1-0716-4578-9_18.
Cystine-knot peptides, with their exceptional thermal stability and resistance to proteolytic degradation, present highly favorable biophysical properties for use as scaffolds in engineering of binders for diverse applications. Multiple variants of knottins with prescribed functional characteristics were described in the literature. These were obtained by rational engineering of natural peptides or by combinatorial library screening. Herein, we describe in detail an ultra-high-throughput screening method of cystine-rich peptide libraries making use of yeast surface display. This is exemplified by converting a trypsin-like protease inhibiting oMCoTI-II cystine-knot framework to a binder of the immune-oncology target CTLA-4. Combinatorial library cloning, followed by yeast surface presentation and sorting, analysis of single clones, and finally chemical synthesis of cystine-knot peptides and their functional validation, provides a robust method for obtaining cystine-rich peptides with novel functional characteristics.
胱氨酸结肽具有出色的热稳定性和抗蛋白水解降解能力，展现出极为有利的生物物理特性，可作为支架用于多种应用的结合物工程。文献中描述了具有特定功能特性的多种结蛋白变体。这些变体通过对天然肽进行理性工程改造或通过组合文库筛选获得。在此，我们详细描述了一种利用酵母表面展示技术对富含胱氨酸的肽文库进行超高通量筛选的方法。以将类胰蛋白酶抑制肽oMCoTI-II的胱氨酸结骨架转化为免疫肿瘤学靶点CTLA-4的结合物为例进行说明。组合文库克隆，随后进行酵母表面展示和分选、单克隆分析，最后对胱氨酸结肽进行化学合成及其功能验证，提供了一种获得具有新功能特性的富含胱氨酸肽的可靠方法。
