Thaiprayoon Apisitt, Chantarasorn Yodpong, Oonanant Worrapoj, Kasorn Anongnard, Longsompurana Phoomintara, Tapaneeyakorn Satita, Riangrungroj Pinpunya, Loison Fabien, Kruse Andrew C, DeLisa Matthew P, Waraho-Zhmayev Dujduan
Biological Engineering Program, Faculty of Engineering, King Mongkut's University of Technology Thonburi, Bangkok, 10140, Thailand.
Division of Ophthalmology, Faculty of Medicine, Vajira Hospital, Navamindradhiraj University, Bangkok, Thailand.
Sci Rep. 2025 Jan 28;15(1):3594. doi: 10.1038/s41598-025-88032-1.
Nanobodies (Nbs) hold great potential to replace conventional antibodies in various biomedical applications. However, conventional methods for their discovery can be time-consuming and expensive. We have developed a reliable protein selection strategy that combines magnetic activated cell sorting (MACS)-based screening of yeast surface display (YSD) libraries and functional ligand-binding identification by Tat-based recognition of associating proteins (FLI-TRAP) to isolate antigen-specific Nbs from synthetic libraries. This combined process enabled isolation of three unique Nb clones (NbT15, NbT21, and NbT22) that all bound specifically to a target antigen, namely proprotein convertase subtilisin/kexin type 9 (PCSK9) as well as a gain-of-function PCSK9 mutant (D374Y). All three clones bound to PCSK9 and blocked the interaction between the low-density lipoprotein receptor (LDLR) and either wild-type PCSK9 or the D374Y mutant. Overall, our combined protein selection method enables rapid and straightforward identification of potent antigen-specific Nbs in a manner that can be executed in a basic laboratory setting without the need for specialized equipment. We anticipate that our strategy will be a valuable addition to the protein engineering toolkit, allowing development of Nbs or virtually any other synthetic binding protein for a wide range of applications.
纳米抗体（Nbs）在各种生物医学应用中具有取代传统抗体的巨大潜力。然而，传统的纳米抗体发现方法可能既耗时又昂贵。我们开发了一种可靠的蛋白质筛选策略，该策略结合了基于磁性激活细胞分选（MACS）的酵母表面展示（YSD）文库筛选以及通过基于Tat的相关蛋白识别进行功能配体结合鉴定（FLI-TRAP），以从合成文库中分离抗原特异性纳米抗体。这一联合过程使得能够分离出三个独特的纳米抗体克隆（NbT15、NbT21和NbT22），它们均能特异性结合靶抗原，即前蛋白转化酶枯草杆菌蛋白酶/kexin 9型（PCSK9）以及一种功能获得性PCSK9突变体（D374Y）。所有这三个克隆均与PCSK9结合，并阻断了低密度脂蛋白受体（LDLR）与野生型PCSK9或D374Y突变体之间的相互作用。总体而言，我们的联合蛋白质筛选方法能够以一种可在基础实验室环境中进行且无需专门设备的方式，快速、直接地鉴定出有效的抗原特异性纳米抗体。我们预计，我们的策略将成为蛋白质工程工具包中的一项有价值的补充，有助于开发用于广泛应用的纳米抗体或几乎任何其他合成结合蛋白。
