Kinetic and molecular insight into immunoglobulin G binding to immobilized recombinant protein A of different orientations.
Kinetic and molecular insight into immunoglobulin G binding to immobilized recombinant protein A of different orientations.
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DOI：
10.1016/j.chroma.2022.463040
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发表时间：
2022-04
期刊：
影响因子：
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通讯作者：
Xinshuang Chu;Xue Yang;Qinghong Shi;Xiaoyan Dong;Y. Sun
中科院分区：
文献类型：
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作者：
Xinshuang Chu;Xue Yang;Qinghong Shi;Xiaoyan Dong;Y. Sun
了解免疫球蛋白G （IgG）与蛋白A结合的机制对高效蛋白A色谱的设计和开发至关重要。本研究通过在金黄色葡萄球菌蛋白A的n端引入半胱氨酸残基（Cys-Z）或在c端引入半胱氨酸-赖氨酸二肽（Z- cys）对其IgG结合域(Z)进行基因修饰，并利用这两种配体通过结合动力学和不同的单分子测量来揭示IgG的结合机制。小鼠骨髓瘤IgG2a （mIgG2a）与两种配体结合动力学的表面等离子体共振（SPR）测量表明，定向配体固定化显著提高了mIgG2a的结合速率常数，并且在三种配体（Cys-Z、Z- cys和Z）中，Z- cys与mIgG2a的结合亲和力最高。这归因于高缔合速率常数和低解离速率常数对mIgG2a的协同作用。此外，带有能量耗散监测（QCM-D）测量的石英晶体微天平提供了igg在z - cys固定芯片上的最大吸附密度，因为igg的zeta电位接近于零。QCM-D研究表明，吸附层依赖于配体类型、密度和IgG。此外，抗原-抗体反应证明，Z-Cys和Cys-Z诱导IgG在翻转方向上结合。最后，引入矩形DNA折纸砖来分析吸附的IgG的分子取向。单分子成像显示，mIgG2a与瓷砖上的柔性Z-Cys主要在侧面和端向上方向相关。该研究为IgG分子在液固界面的结合机制提供了分子视角，并将有助于设计新的基于蛋白质的配体和高容量吸附剂。
Mechanistic understanding of immunoglobulin G (IgG) binding to protein A is crucial for the design and development of high-performance protein A chromatography. In this work, the IgG binding domain (Z) of protein A fromStaphylococcus aureuswas genetically modified by introducing a cysteine residue at the N-terminus (Cys-Z) or a cysteine-lysine dipeptide at the C-terminus (Z-Cys), and the two ligands were used to unravel the IgG binding mechanism by means of binding kinetics and different single molecule measurements. Surface plasma resonance (SPR) measurement of the binding kinetics of mouse myeloma IgG2a (mIgG2a) to the two ligands indicated that oriented ligand immobilization significantly increased the association rate constant of mIgG2a, and Z-Cys had the highest binding affinity to mIgG2a among the three ligands (Cys-Z, Z-Cys and Z). This was attributed to the synergistic contribution of the high association rate constant and low dissociation rate constant to mIgG2a. Furthermore, quartz crystal microbalance with energy dissipation monitoring (QCM-D) measurement provided the maximum adsorption densities of IgGs on the Z-Cys-immobilized chip as zeta potentials of IgGs were nearly zero. The QCM-D investigation revealed that the adsorbed layer was dependent on ligand type and density, and IgG. Moreover, Z-Cys and Cys-Z induced IgG binding in flipped orientations, as evidenced by the antigen-antibody reaction. Finally, rectangular DNA origami tiles were introduced to analyze the molecular orientation of adsorbed IgG. Single-molecule imaging showed that mIgG2a was associated with flexible Z-Cys on the tiles predominantly in side-on and end-on orientations. The research has provided molecular insight into the binding mechanism of IgG molecules at liquid–solid interfaces and would help design new protein A-based ligands and high-capacity adsorbents.