Journal Article

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

Chantarasorn, Yodpong, Longsompurana, Phoomintara, Tapaneeyakorn, Satita, Kasorn, Anongnard, Kruse, Andrew C., Loison, Fabien, Oonanant, Worrapoj, Waraho-Zhmayev, Dujduan, DeLisa, Matthew P., Thaiprayoon, Apisitt, Riangrungroj, Pinpunya

2025

Overview

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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 631/154 / 631/1647/1511 / 631/1647/2163 / 631/1647/2234 / 631/1647/338 / 631/337 / 631/45 / Antigens / Camelid antibody / Genetic selection / High-throughput screening / Humanities and Social Sciences / Humans / Hypercholesterolemia / Kexin / Libraries / multidisciplinary / Mutants / Nanobodies / Peptide Library / Proprotein Convertase 9 - genetics / Proprotein Convertase 9 - immunology / Proprotein Convertase 9 - metabolism / Proprotein convertases / Protein Binding / Protein engineering / Protein transport / Proteins / Receptor density / Receptors, LDL - metabolism / Science / Science (multidisciplinary) / Single-Domain Antibodies - genetics / Single-Domain Antibodies - immunology / Single-Domain Antibodies - isolation & purification / Single-Domain Antibodies - metabolism / Subtilisin / Synthetic antibody library / VHH single-domain antibody / Yeast