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

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

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 s...

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Main Authors: Apisitt Thaiprayoon, Yodpong Chantarasorn, Worrapoj Oonanant, Anongnard Kasorn, Phoomintara Longsompurana, Satita Tapaneeyakorn, Pinpunya Riangrungroj, Fabien Loison, Andrew C. Kruse, Matthew P. DeLisa, Dujduan Waraho-Zhmayev
Format: Article
Language:English
Published: Nature Portfolio 2025-01-01
Series:Scientific Reports
Subjects:
Camelid antibody
Genetic selection
High-throughput screening
Hypercholesterolemia
Synthetic antibody library
VHH single-domain antibody
Online Access:https://doi.org/10.1038/s41598-025-88032-1
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Summary: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.
ISSN:2045-2322

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