screening of PCSK9 specific nanobody by combination of FLI-TRAP and yeast display
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 wild-type (wt) proprotein convertase subtilisin/kexin type 9 (PCSK9) as well as a gain-of-function PCSK9 mutant (D374Y). Clone NbT21 exhibited the strongest binding affinity with an equilibrium dissociation constant (Kd) of 1 nM for its interaction with wt PCSK9. Clones NbT15 and NbT22 exhibted Kd values of 38 and 30 nM, respectively. Additionally, NbT15, NbT21, and NbT22 blocked the interaction between the low-density lipoprotein receptor (LDLR) and either wt PCSK9 or the D374Y mutant. Molecular docking revealed that NbT21 binds PCSK9 near the epidermal growth factor-like repeat A binding domain, while NbT15 and NbT21 showed strong binding tendencies with the catalytic domain cavity. 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.
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 wild-type (wt) proprotein convertase subtilisin/kexin type 9 (PCSK9) as well as a gain-of-function PCSK9 mutant (D374Y). Clone NbT21 exhibited the strongest binding affinity with an equilibrium dissociation constant (Kd) of 1 nM for its interaction with wt PCSK9. Clones NbT15 and NbT22 exhibted Kd values of 38 and 30 nM, respectively. Additionally, NbT15, NbT21, and NbT22 blocked the interaction between the low-density lipoprotein receptor (LDLR) and either wt PCSK9 or the D374Y mutant. Molecular docking revealed that NbT21 binds PCSK9 near the epidermal growth factor-like repeat A binding domain, while NbT15 and NbT21 showed strong binding tendencies with the catalytic domain cavity. 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.
king mongkut's university of technology thonburi