The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface
The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface
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蛋白 A 色谱中的珍珠项链模型：树脂界面的分子机制
DOI：
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发表时间：
2018
影响因子：
3.8
通讯作者：
R. Tscheliessnig
中科院分区：
文献类型：
--
作者：
Goncalo L Silva;Jacek Plewka;H. Lichtenegger;A. C. Dias;A. Jungbauer;R. Tscheliessnig
葡萄球菌蛋白 A 层析是下游处理中单克隆抗体纯化和捕获的成熟核心技术。 MabSelect SuRe 涉及葡萄球菌蛋白 A 的 B 结构域（称为 Z 结构域）重组形式的四聚体链。关于化学计量、结合方向或优选结合知之甚少。我们分析了在不同抗体浓度下固定在工业高度相关的色谱树脂中的抗体-蛋白 A 复合物的小角 X 射线散射数据。根据散射数据，我们计算了归一化径向密度分布。我们设计了三维 (3D) 模型，其中包含 IgG1（此处使用的曲妥珠单抗同种型；蛋白质数据库：1HZH）和葡萄球菌蛋白 A B 结构域（MabSelect SuRe 树脂中包含的重组结构的天然形式；蛋白质数据库：1BDD）的蛋白质数据库晶体结构。我们计算了不同抗体与 A 蛋白化学计量（1:1、2:1 和 3:1）的不同结合构象，并将根据 3D 模型计算的归一化径向密度分布与从实验数据获得的分布进行比较。在等温线的线性范围内，我们倾向于 1:1 的比例，抗体以非常低和高的浓度结合到蛋白 A 链的外部结构域。在饱和区，更可能出现 2:1 的比率。由于空间效应，3:1 化学计量被排除。
Staphylococcal protein A chromatography is an established core technology for monoclonal antibody purification and capture in the downstream processing. MabSelect SuRe involves a tetrameric chain of a recombinant form of the B domain of staphylococcal protein A, called the Z‐domain. Little is known about the stoichiometry, binding orientation, or preferred binding. We analyzed small‐angle X‐ray scattering data of the antibody–protein A complex immobilized in an industrial highly relevant chromatographic resin at different antibody concentrations. From scattering data, we computed the normalized radial density distributions. We designed three‐dimensional (3D) models with protein data bank crystallographic structures of an IgG1 (the isoform of trastuzumab, used here; Protein Data Bank: 1HZH) and the staphylococcal protein A B domain (the native form of the recombinant structure contained in MabSelect SuRe resin; Protein Data Bank: 1BDD). We computed different binding conformations for different antibody to protein A stoichiometries (1:1, 2:1, and 3:1) and compared the normalized radial density distributions computed from 3D models with those obtained from the experimental data. In the linear range of the isotherm we favor a 1:1 ratio, with the antibody binding to the outer domains in the protein A chain at very low and high concentrations. In the saturation region, a 2:1 ratio is more likely to occur. A 3:1 stoichiometry is excluded because of steric effects.
DOI：
10.1073/pnas.97.10.5399
发表时间：
2000-05-09
影响因子：
11.1
作者：
Graille, M;Stura, EA;Silverman, GJ
通讯作者：
Silverman, GJ
影响因子：
3.4
作者：
Brandt, J. Paul;Patapoff, Thomas W.;Aragon, Sergio R.
通讯作者：
Aragon, Sergio R.