Interface-Based Design of High-Affinity Affibody Ligands for the Purification of RBD from Spike Proteins
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Published:2023-08-30
Issue:17
Volume:28
Page:6358
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ISSN:1420-3049
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Container-title:Molecules
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language:en
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Short-container-title:Molecules
Author:
Song Siyuan1,
Shi Qinghong12
Affiliation:
1. Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China
2. Key Laboratory of Systems Bioengineering and Frontiers Science Center for Synthetic Biology (Ministry of Education), Tianjin University, Tianjin 300350, China
Abstract
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.
Funder
National Key Research and Development Program of China
National Natural Science Foundation of China
Foundation for Innovative Research Groups of the National Natural Science Foundation of China
Subject
Chemistry (miscellaneous),Analytical Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Molecular Medicine,Drug Discovery,Pharmaceutical Science