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Insights into the molecular structure of immobilized protein A ligands on dextran-coated nanoparticles: Comprehensive spectroscopic investigation
- Wang, Zhen, Shen, Yi, Shi, Qing-Hong, Sun, Yan
- Biochemical engineering journal 2019 v.146 pp. 20-30
- buffer index, buffers, calorimetry, chemical structure, chromatography, dextran, electrostatic interactions, humans, immunoglobulin G, ligands, magnetism, nanoparticles, pH, polymerization, spectral analysis
- Ligand immobilization is indeed key fundamental for the development of high-capacity protein A chromatographic material. In this work, mono- and tetra-mers of Z domain from protein A were immobilized onto magnetic nanoparticle (NP) coated with carboxymethyl dextran (CMD), Fe3O4@CMD NP, via oriented coupling. Spectral results of Fe3O4@CMD-oZ NP with oriented Z domain showed that the molecular structure of the ligand was dominated by ligand – surface interaction. At pH 4.5, the ligand was disrupted by electrostatic attraction between NP surface and ligand. As buffer pH was higher than pI of the ligand, the ligand tended to be native structure with an increase of electrostatic repulsion between NP surface and ligands. At pH 10.0, therefore, the ligand remained intact on Fe3O4@CMD-oZ NP. Furthermore, ligand polymerization promoted the structural stability of the tetrameric ligand by decreasing interaction between NP surface and three domains at amino terminus of the ligand. It was more significant at pH 7.0 and the highest stoichiometric number of human IgG binding was obtained. Moreover, oriented immobilization was more beneficial for structural stability of the tetrameric ligand than random immobilization. Likely, spectral and calorimetric evidences at different ligand densities further demonstrated that the crowding effect induced at high ligand density maintained molecular structure of the tetrameric ligand better.