Thaiprayoon A.Chantarasorn Y.Oonanant W.Kasorn A.Longsompurana P.Tapaneeyakorn S.Riangrungroj P.Loison F.Kruse A.C.DeLisa M.P.Waraho-Zhmayev D.Mahidol University2025-02-172025-02-172025-01-28Scientific reports Vol.15 No.1 (2025) , 3594https://repository.li.mahidol.ac.th/handle/20.500.14594/105333Nanobodies (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.MultidisciplinaryArticleSCOPUS10.1038/s41598-025-88032-12-s2.0-852172737802045232239875480