Isolation of PCSK9-specific nanobodies from synthetic libraries using a combined protein selection strategy

Journal article

Authors/Editors

DUJDUAN WARAHO

Strategic Research Themes

Medical Treatment & Prevention (Smart Healthcare)

Publication Details

Author list: ApisittThaiprayoon, Yodpong Chantarasorn, Worrapoj Oonanant, Anongnard Kasorn, Phoomintara Longsompurana, SatitaTapaneeyakorn4, Pinpunya Riangrungroj,Fabien Loison, Andrew C. Kruse, Matthew P. DeLisa & Dujduan Waraho-Zhmayev

Publisher: Nature Research

Publication year: 2025

Journal: Scientific Reports (2045-2322)

Volume number: 15

Issue number: 1

ISSN: 2045-2322

eISSN: 2045-2322

Languages: English-Great Britain (EN-GB)

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Abstract

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.

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