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

• Published in: Molecules (Basel, Switzerland) Vol. 28; no. 17; p. 6358
• Main Authors: Song, Siyuan, Shi, Qinghong
• Format: Journal Article
• Language: English
• Published: Basel MDPI AG 30.08.2023; MDPI
• Subjects: affibody; affinity chromatography; binding affinity; Chromatography; Coronaviruses; COVID-19 vaccines; Energy; Health aspects; Libraries; ligand design; Ligands; Manufacturing; molecular dynamics simulation; Mutation; Peptides; Proteins; Reagents; receptor binding domain; Severe acute respiratory syndrome; Severe acute respiratory syndrome coronavirus 2; Simulation; Vaccines; Viruses; China
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules28176358

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 ZRBD affibody ligands. Molecular docking was performed using HADDOCK software, and three potential ZRBD affibodies, ZRBD-02, ZRBD-04, and ZRBD-07, were obtained. Molecular dynamics (MD) simulation verified that the binding of ZRBD 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 ZRBD affibodies had high affinity for RBD binding, and the lowest dissociation constant was 36.3 nmol/L for ZRBD-07 among the three potential ZRBD affibodies. Herein, ZRBD-02 and ZRBD-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 ZRBD SepFF gels and was eluted by 0.1 mol/L NaOH. Moreover, the ZRBD-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.