Interface-Based Design of High-Affinity Affibody Ligands for the Purification of RBD from Spike Proteins

The outbreak of coronavirus disease 2019 (COVID-19) has sparked an urgent demand for advanced diagnosis and vaccination worldwide. The discovery of high-affinity ligands is of great significance for vaccine and diagnostic reagent manufacturing. Targeting the receptor binding domain (RBD) from the spike protein of severe acute respiratory syndrome-coronavirus 2, an interface at the outer surface of helices on the Z domain from protein A was introduced to construct a virtual library for the screening of Z_RBD affibody ligands. Molecular docking was performed using HADDOCK software, and three potential Z_RBD affibodies, Z_RBD-02, Z_RBD-04, and Z_RBD-07, were obtained. Molecular dynamics (MD) simulation verified that the binding of Z_RBD affibodies to RBD was driven by electrostatic interactions. Per-residue free energy decomposition analysis further substantiated that four residues with negative-charge characteristics on helix α1 of the Z domain participated in this process. Binding affinity analysis by microscale thermophoresis showed that Z_RBD affibodies had high affinity for RBD binding, and the lowest dissociation constant was 36.3 nmol/L for Z_RBD-07 among the three potential Z_RBD affibodies. Herein, Z_RBD-02 and Z_RBD-07 affibodies were selected for chromatographic verifications after being coupled to thiol-activated Sepharose 6 Fast Flow (SepFF) gel. Chromatographic experiments showed that RBD could bind on both Z_RBD SepFF gels and was eluted by 0.1 mol/L NaOH. Moreover, the Z_RBD-07 SepFF gel had a higher affinity for RBD. This research provided a new idea for the design of affibody ligands and validated the potential of affibody ligands in the application of RBD purification from complex feedstock.