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 subst...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 Fe_(3)O_(4) magnetic nanoparticles(Fe_(3)O_(4)@Dx MNPs), affinity MNPs with the immobilized ligand via N11C on Helix Ⅰ(Fe_(3)O_(4)@Dx-Z_(1) MNPs) had the highest helical content, and MNPs with the immobilized ligand via G29C on Helix Ⅱ(Fe_(3)O_(4)@Dx-Z_(2) MNPs) had the lowest helical content at the same pHs. It was attributed to less electrostatic attraction of ligand to negatively charged surface on Fe_(3)O_(4)@Dx-Z_(1) 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 Fe_(3)O_(4)@Dx-Z_(1) MNPs in isothermal titration calorimetry measurement, further validating greater structural integrity of the ligand on Fe_(3)O_(4)@Dx-Z_(1) 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.展开更多
基金supported by the National Natural Science Foundation of China (Nos. 21878221 and 21476166)the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (No. 21621004)。
文摘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 Fe_(3)O_(4) magnetic nanoparticles(Fe_(3)O_(4)@Dx MNPs), affinity MNPs with the immobilized ligand via N11C on Helix Ⅰ(Fe_(3)O_(4)@Dx-Z_(1) MNPs) had the highest helical content, and MNPs with the immobilized ligand via G29C on Helix Ⅱ(Fe_(3)O_(4)@Dx-Z_(2) MNPs) had the lowest helical content at the same pHs. It was attributed to less electrostatic attraction of ligand to negatively charged surface on Fe_(3)O_(4)@Dx-Z_(1) 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 Fe_(3)O_(4)@Dx-Z_(1) MNPs in isothermal titration calorimetry measurement, further validating greater structural integrity of the ligand on Fe_(3)O_(4)@Dx-Z_(1) 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.
