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
Inspired by nature,precise spatial organization of enzyme cascades of interest is crucial to the improvement of catalytic performance.Herein,DNA scaffolds were introduced to construct a toolkit for versatile immobiliz...Inspired by nature,precise spatial organization of enzyme cascades of interest is crucial to the improvement of catalytic performance.Herein,DNA scaffolds were introduced to construct a toolkit for versatile immobilization of enzyme pairs on dextran-coated magnetic nanoparticles(MNPs).After the glucose oxidase(GOx)and horseradish peroxidase(HRP)pair was immobilized through random cova-lent,DNA-directed and DNA tile-directed strategies,the immobilized GOx/HRP pair on the MNP-based carrier assembled with DNA tile(TD@MNPs)exhibited the highest activity due to rational spatial organization and less conformational change of constituent enzymes.With a decrease in interenzyme distance on TD@MNPs,furthermore,the catalytic efficiency of the HRP/GOx pair increased further for both substrates,2,2'-azinobis(3-ethyl-benzthiazoline-6-sulfonate)(ABTS)and 3,3',5,5'-tetramethyl benzidine(TMB).As the assembled HRP was closer to the carrier surface,the catalytic efficiency of the GOx/HRP pair increased by 6.2-fold for positively charged TMB and only by 62%for negatively charged ABTS compared with the free GOx/HRP pair.Moreover,a reversal of catalytic efficiency was found after the GOx/HRP pair was assembled on a positively charged carrier(TD@pMNPs).This research demonstrated that MNP-based car-riers had the potential to become a versatile toolkit for shedding an insight into catalytic performance and the development of new biocatalysts.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.
基金the National Key Research and Development Program of China(No.2021YFC2102801)the National Natural Science Foundation of China(No.21878221)the Foundation for Innovative Research Groups of the National Natural Science Foundation of China(No.21621004).
文摘Inspired by nature,precise spatial organization of enzyme cascades of interest is crucial to the improvement of catalytic performance.Herein,DNA scaffolds were introduced to construct a toolkit for versatile immobilization of enzyme pairs on dextran-coated magnetic nanoparticles(MNPs).After the glucose oxidase(GOx)and horseradish peroxidase(HRP)pair was immobilized through random cova-lent,DNA-directed and DNA tile-directed strategies,the immobilized GOx/HRP pair on the MNP-based carrier assembled with DNA tile(TD@MNPs)exhibited the highest activity due to rational spatial organization and less conformational change of constituent enzymes.With a decrease in interenzyme distance on TD@MNPs,furthermore,the catalytic efficiency of the HRP/GOx pair increased further for both substrates,2,2'-azinobis(3-ethyl-benzthiazoline-6-sulfonate)(ABTS)and 3,3',5,5'-tetramethyl benzidine(TMB).As the assembled HRP was closer to the carrier surface,the catalytic efficiency of the GOx/HRP pair increased by 6.2-fold for positively charged TMB and only by 62%for negatively charged ABTS compared with the free GOx/HRP pair.Moreover,a reversal of catalytic efficiency was found after the GOx/HRP pair was assembled on a positively charged carrier(TD@pMNPs).This research demonstrated that MNP-based car-riers had the potential to become a versatile toolkit for shedding an insight into catalytic performance and the development of new biocatalysts.
