High-throughput enrichment of functional disulfide-rich peptides by droplet microfluidics†

IF 5.4 2区 工程技术 Q1 BIOCHEMICAL RESEARCH METHODS
Lab on a Chip Pub Date : 2025-06-04 DOI:10.1039/D5LC00261C
Jing Xie, Kuok Yap, Simon J. de Veer, Selvakumar Edwardraja, Thomas Durek, Matt Trau, David J. Craik and Conan K. Wang
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引用次数: 0

Abstract

Disulfide-rich peptides (DRPs) have evolved intricate topologies to carry out a wide range of bioactivities throughout nature, e.g., in fungi, insects, plants and animals, and have proven applications in medicine and agriculture. To discover novel DRPs, it is now routine to screen DRP libraries for target affinity, but target binding does not necessarily correlate with function. This study reports an innovative platform for screening of DRP libraries based on the functional endpoint of biochemical reactions within picoliter-sized water-in-oil droplets. We leveraged yeast secretory expression to ensure proper assembly of disulfide connectivity, and thus peptide shape, and engineered customizable strains for facile detection of function (i.e., protease inhibitory activity) for libraries of DRPs. Rapid enrichment of a potent trypsin inhibitor (MCoTI-II) from a >100 000 pool of randomized variants across four rounds of selection was achieved, far exceeding the library sizes explored previously for peptide systems in droplet microfluidics. This developed platform provides a foundation to explore the functional engineering of DRPs.

Abstract Image

液滴微流控技术高通量富集功能性富二硫肽
富二硫肽(DRPs)已经进化出复杂的拓扑结构,在自然界中进行广泛的生物活性,例如真菌、昆虫、植物和动物,并已在医学和农业中得到证实。为了发现新的DRP,现在筛选DRP库的目标亲和力是常规的,但目标结合并不一定与功能相关。本研究报告了一个基于皮升大小的油包水滴内生化反应功能终点的DRP文库筛选的创新平台。我们利用酵母分泌表达来确保二硫化物连接的正确组装,从而确保肽的形状,并设计了可定制的菌株,以便于检测DRPs文库的功能(即蛋白酶抑制活性)。通过4轮筛选,从100,000个随机变异库中快速富集了一种强效胰蛋白酶抑制剂(MCoTI-II),远远超过了之前在微滴微流体中探索的肽系统的文库大小。该开发平台为探索DRPs的功能工程提供了基础。
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来源期刊
Lab on a Chip
Lab on a Chip 工程技术-化学综合
CiteScore
11.10
自引率
8.20%
发文量
434
审稿时长
2.6 months
期刊介绍: Lab on a Chip is the premiere journal that publishes cutting-edge research in the field of miniaturization. By their very nature, microfluidic/nanofluidic/miniaturized systems are at the intersection of disciplines, spanning fundamental research to high-end application, which is reflected by the broad readership of the journal. Lab on a Chip publishes two types of papers on original research: full-length research papers and communications. Papers should demonstrate innovations, which can come from technical advancements or applications addressing pressing needs in globally important areas. The journal also publishes Comments, Reviews, and Perspectives.
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