Unraveling structure and performance of protein a ligands at liquid–solid interfaces: A multi-techniques analysis

doi:10.1016/j.cjche.2021.09.027

中国化学工程学报 ›› 2023, Vol. 54 ›› Issue (2): 232-239.DOI: 10.1016/j.cjche.2021.09.027

• Full Length Article • 上一篇下一篇

Unraveling structure and performance of protein a ligands at liquid–solid interfaces: A multi-techniques analysis

Yi Shen¹, Xinshuang Chu¹, Qinghong Shi^1,2

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

收稿日期:2021-08-06 修回日期:2021-09-13 出版日期:2023-02-28 发布日期:2023-05-11
通讯作者: Qinghong Shi,E-mail:qhshi@tju.edu.cn
基金资助:
This work was supported by the National Natural Science Foundation of China (Nos. 21878221 and 21476166), and the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (No. 21621004).

Unraveling structure and performance of protein a ligands at liquid–solid interfaces: A multi-techniques analysis

Yi Shen¹, Xinshuang Chu¹, Qinghong Shi^1,2

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

Received:2021-08-06 Revised:2021-09-13 Online:2023-02-28 Published:2023-05-11
Contact: Qinghong Shi,E-mail:qhshi@tju.edu.cn
Supported by:
This work was supported by the National Natural Science Foundation of China (Nos. 21878221 and 21476166), and the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (No. 21621004).

摘要/Abstract

摘要： Oriented ligand immobilization is one of the most effective strategies used in the design and construction of a high-capacity protein A chromatography. In this work, cysteine was introduced as anchoring sites by substituting a specific residue on Helix I, II, and at C-terminus of antibody binding domain Z from protein A, respectively, to investigate structural evolution and binding behavior of protein A ligands at liquid–solid interfaces. Among the three affinity dextran-coated Fe₃O₄ magnetic nanoparticles (Fe₃O₄@Dx MNPs), affinity MNPs with the immobilized ligand via N11C on Helix I (Fe₃O₄@Dx-Z₁ MNPs) had the highest helical content, and MNPs with the immobilized ligand via G29C on Helix II (Fe₃O₄@Dx-Z₂ MNPs) had the lowest helical content at the same pHs. It was attributed to less electrostatic attraction of ligand to negatively charged surface on Fe₃O₄@Dx-Z₁ MNPs because of less positive charged residues on Helix I (K6) than Helix II (R27/K35). Among the three affinity MNPs, moreover, the highest affinity to immunoglobulin G (IgG) binding was observed on Fe₃O₄@Dx-Z₁ MNPs in isothermal titration calorimetry measurement, further validating greater structural integrity of the ligand on Fe₃O₄@Dx-Z₁ MNPs. Finally, the study of IgG binding on MNPs and 96-well plates showed that anchoring sites for ligand immobilization had distinct influences on IgG binding and IgG-mediated antigen binding. This work illustrated that anchoring sites of the ligands had a striking significance for the molecular structure of the ligand at liquid–solid interfaces and raised an important implication for the design and optimization of protein A chromatography and protein A-based immunoassay analysis.

关键词: Adsorption, Interface, Thermodynamics, Protein A ligand, Immobilization, Molecular structure

Abstract: Oriented ligand immobilization is one of the most effective strategies used in the design and construction of a high-capacity protein A chromatography. In this work, cysteine was introduced as anchoring sites by substituting a specific residue on Helix I, II, and at C-terminus of antibody binding domain Z from protein A, respectively, to investigate structural evolution and binding behavior of protein A ligands at liquid–solid interfaces. Among the three affinity dextran-coated Fe₃O₄ magnetic nanoparticles (Fe₃O₄@Dx MNPs), affinity MNPs with the immobilized ligand via N11C on Helix I (Fe₃O₄@Dx-Z₁ MNPs) had the highest helical content, and MNPs with the immobilized ligand via G29C on Helix II (Fe₃O₄@Dx-Z₂ MNPs) had the lowest helical content at the same pHs. It was attributed to less electrostatic attraction of ligand to negatively charged surface on Fe₃O₄@Dx-Z₁ MNPs because of less positive charged residues on Helix I (K6) than Helix II (R27/K35). Among the three affinity MNPs, moreover, the highest affinity to immunoglobulin G (IgG) binding was observed on Fe₃O₄@Dx-Z₁ MNPs in isothermal titration calorimetry measurement, further validating greater structural integrity of the ligand on Fe₃O₄@Dx-Z₁ MNPs. Finally, the study of IgG binding on MNPs and 96-well plates showed that anchoring sites for ligand immobilization had distinct influences on IgG binding and IgG-mediated antigen binding. This work illustrated that anchoring sites of the ligands had a striking significance for the molecular structure of the ligand at liquid–solid interfaces and raised an important implication for the design and optimization of protein A chromatography and protein A-based immunoassay analysis.

Key words: Adsorption, Interface, Thermodynamics, Protein A ligand, Immobilization, Molecular structure

Yi Shen, Xinshuang Chu, Qinghong Shi. Unraveling structure and performance of protein a ligands at liquid–solid interfaces: A multi-techniques analysis[J]. 中国化学工程学报, 2023, 54(2): 232-239.

Yi Shen, Xinshuang Chu, Qinghong Shi. Unraveling structure and performance of protein a ligands at liquid–solid interfaces: A multi-techniques analysis[J]. Chinese Journal of Chemical Engineering, 2023, 54(2): 232-239.

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Unraveling structure and performance of protein a ligands at liquid–solid interfaces: A multi-techniques analysis

Unraveling structure and performance of protein a ligands at liquid–solid interfaces: A multi-techniques analysis

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