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...
Saved in:
| Main Authors: | , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2025-01-01
|
| Series: | Scientific Reports |
| Subjects: | |
| Online Access: | https://doi.org/10.1038/s41598-025-88032-1 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1832571781126291456 |
|---|---|
| author | 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 |
| author_facet | 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 |
| author_sort | Apisitt Thaiprayoon |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-3ccda0e69ac942d3a9d2c92b7dc0447d |
| institution | Kabale University |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-3ccda0e69ac942d3a9d2c92b7dc0447d2025-02-02T12:19:14ZengNature PortfolioScientific Reports2045-23222025-01-0115111410.1038/s41598-025-88032-1Isolation of PCSK9-specific nanobodies from synthetic libraries using a combined protein selection strategyApisitt Thaiprayoon0Yodpong Chantarasorn1Worrapoj Oonanant2Anongnard Kasorn3Phoomintara Longsompurana4Satita Tapaneeyakorn5Pinpunya Riangrungroj6Fabien Loison7Andrew C. Kruse8Matthew P. DeLisa9Dujduan Waraho-Zhmayev10Biological Engineering Program, Faculty of Engineering, King Mongkut’s University of Technology ThonburiDivision of Ophthalmology, Faculty of Medicine, Vajira Hospital, Navamindradhiraj UniversityDepartment of Basic Medical Science, Faculty of Medicine Vajira Hospital, Navamindradhiraj UniversityDepartment of Basic Medical Science, Faculty of Medicine Vajira Hospital, Navamindradhiraj UniversityBiological Engineering Program, Faculty of Engineering, King Mongkut’s University of Technology ThonburiNational Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA)National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and TechnologyDevelopment Agency (NSTDA)Department of Microbiology, Faculty of Science, Mahidol UniversityDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical SchoolRobert F. Smith School of Chemical and Biomolecular Engineering, Cornell UniversityBiological Engineering Program, Faculty of Engineering, King Mongkut’s University of Technology ThonburiAbstract 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.https://doi.org/10.1038/s41598-025-88032-1Camelid antibodyGenetic selectionHigh-throughput screeningHypercholesterolemiaSynthetic antibody libraryVHH single-domain antibody |
| spellingShingle | 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 Isolation of PCSK9-specific nanobodies from synthetic libraries using a combined protein selection strategy Scientific Reports Camelid antibody Genetic selection High-throughput screening Hypercholesterolemia Synthetic antibody library VHH single-domain antibody |
| title | Isolation of PCSK9-specific nanobodies from synthetic libraries using a combined protein selection strategy |
| title_full | Isolation of PCSK9-specific nanobodies from synthetic libraries using a combined protein selection strategy |
| title_fullStr | Isolation of PCSK9-specific nanobodies from synthetic libraries using a combined protein selection strategy |
| title_full_unstemmed | Isolation of PCSK9-specific nanobodies from synthetic libraries using a combined protein selection strategy |
| title_short | Isolation of PCSK9-specific nanobodies from synthetic libraries using a combined protein selection strategy |
| title_sort | isolation of pcsk9 specific nanobodies from synthetic libraries using a combined protein selection strategy |
| topic | Camelid antibody Genetic selection High-throughput screening Hypercholesterolemia Synthetic antibody library VHH single-domain antibody |
| url | https://doi.org/10.1038/s41598-025-88032-1 |
| work_keys_str_mv | AT apisittthaiprayoon isolationofpcsk9specificnanobodiesfromsyntheticlibrariesusingacombinedproteinselectionstrategy AT yodpongchantarasorn isolationofpcsk9specificnanobodiesfromsyntheticlibrariesusingacombinedproteinselectionstrategy AT worrapojoonanant isolationofpcsk9specificnanobodiesfromsyntheticlibrariesusingacombinedproteinselectionstrategy AT anongnardkasorn isolationofpcsk9specificnanobodiesfromsyntheticlibrariesusingacombinedproteinselectionstrategy AT phoomintaralongsompurana isolationofpcsk9specificnanobodiesfromsyntheticlibrariesusingacombinedproteinselectionstrategy AT satitatapaneeyakorn isolationofpcsk9specificnanobodiesfromsyntheticlibrariesusingacombinedproteinselectionstrategy AT pinpunyariangrungroj isolationofpcsk9specificnanobodiesfromsyntheticlibrariesusingacombinedproteinselectionstrategy AT fabienloison isolationofpcsk9specificnanobodiesfromsyntheticlibrariesusingacombinedproteinselectionstrategy AT andrewckruse isolationofpcsk9specificnanobodiesfromsyntheticlibrariesusingacombinedproteinselectionstrategy AT matthewpdelisa isolationofpcsk9specificnanobodiesfromsyntheticlibrariesusingacombinedproteinselectionstrategy AT dujduanwarahozhmayev isolationofpcsk9specificnanobodiesfromsyntheticlibrariesusingacombinedproteinselectionstrategy |