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

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. (c) 2022 The Chemical Industry and Engineering Society of China, and Chemical Industry Press Co., Ltd. All rights reserved.

Automated summary

This study investigated the structural evolution and binding behavior of protein A ligands at liquid-solid interfaces by introducing cysteine as anchoring sites. It was found that the ligands immobilized through the anchoring site on Helix I exhibited the highest helical content and affinity to immunoglobulin G (IgG) binding. The study also revealed the significant influence of anchoring sites on the molecular structure of the ligands and IgG-mediated antigen binding.