Isolation of PCSK9-specific nanobodies from synthetic libraries using a combined protein selection strategy
Issued Date
2025-01-28
Resource Type
eISSN
20452322
Scopus ID
2-s2.0-85217273780
Pubmed ID
39875480
Journal Title
Scientific reports
Volume
15
Issue
1
Rights Holder(s)
SCOPUS
Bibliographic Citation
Scientific reports Vol.15 No.1 (2025) , 3594
Suggested Citation
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. Isolation of PCSK9-specific nanobodies from synthetic libraries using a combined protein selection strategy. Scientific reports Vol.15 No.1 (2025) , 3594. doi:10.1038/s41598-025-88032-1 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/105333
Title
Isolation of PCSK9-specific nanobodies from synthetic libraries using a combined protein selection strategy
Author's Affiliation
Cornell Institute of Biotechnology
Faculty of Science, Mahidol University
Cornell University College of Engineering
Vajira Hospital
Thailand National Nanotechnology Center
Thailand National Center for Genetic Engineering and Biotechnology
King Mongkut's University of Technology Thonburi
Harvard Medical School
Faculty of Science, Mahidol University
Cornell University College of Engineering
Vajira Hospital
Thailand National Nanotechnology Center
Thailand National Center for Genetic Engineering and Biotechnology
King Mongkut's University of Technology Thonburi
Harvard Medical School
Corresponding Author(s)
Other Contributor(s)
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.