Styles Matthew J, Pixley Joshua A, Wei Tongyao, Basile Christopher, Lu Shannon S, Dickinson Bryan C
Department of Chemistry, University of Chicago, Chicago, IL, USA.
Chan Zuckerberg Biohub, Chicago, IL, USA.
Nat Methods. 2025 Aug;22(8):1720-1730. doi: 10.1038/s41592-025-02740-0. Epub 2025 Aug 6.
Proteins that selectively bind to a target of interest are foundational in research, diagnostics and therapeutics. Current approaches for discovering binders are laborious and time-consuming, taking months or more, and have a high failure rate. Here we establish phage-assisted noncontinuous selection of protein binders (PANCS-Binders), an in vivo selection platform that links the life cycle of M13 phage to target protein binding through proximity-dependent split RNA polymerase biosensors, allowing for comprehensive screening of whether a variant binds a target with high fidelity. We showcase PANCS-Binders by screening multiple protein libraries each against a panel of 95 separate targets, thereby individually assessing more than 10 protein-protein interaction pairs, in 2 days. These selections yielded large, high-quality datasets and hundreds of novel binders, which can be affinity matured or directly used in mammalian cells to inhibit or degrade targets. We believe that PANCS-Binders accelerates and simplifies the binder discovery process, which will help unlock new creative potential in proteome targeting with engineered binder-based biotechnologies.
能够选择性结合目标蛋白的蛋白质在研究、诊断和治疗领域具有重要意义。目前发现结合蛋白的方法既费力又耗时，通常需要数月甚至更长时间，而且失败率很高。在此，我们建立了噬菌体辅助的蛋白质结合剂非连续筛选方法（PANCS-Binders），这是一种体内筛选平台，它通过依赖于邻近性的分裂RNA聚合酶生物传感器将M13噬菌体的生命周期与目标蛋白结合联系起来，从而能够全面筛选变体是否能高保真地结合目标。我们通过针对95个不同目标的组合筛选多个蛋白质文库来展示PANCS-Binders，从而在两天内单独评估超过10个蛋白质-蛋白质相互作用对。这些筛选产生了大量高质量的数据集和数百种新型结合剂，这些结合剂可以进行亲和力成熟处理，或直接用于哺乳动物细胞中以抑制或降解目标。我们相信，PANCS-Binders加速并简化了结合剂的发现过程，这将有助于利用基于工程化结合剂的生物技术在蛋白质组靶向中释放新的创新潜力。
