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Characterization and Design of Chemically Selective Cationic Displacers Using a Robotic High-Throughput Screen

机译：使用机器人高通量筛的化学选择性阳离子置换剂的表征和设计

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A robotic high-throughput displacer screen was developed and employed to identify chemically selective displacers for several protein pairs in cation exchange chromatography. This automated screen enabled the evaluation of a wide range of experimental conditions in a relatively short period of time. Displacers were evaluated at multiple concentrations for these protein pairs,and DC-50 plots were constructed. Selectivity pathway plots were also constructed and different regimes were established for selective and exclusive separations. Importantly,selective displacement was found to be conserved for multiple protein pairs,demonstrating the technique to be applicable for a range of protein systems. Although chemically selective displacers were able to separate protein pairs that had similar retention in ion exchange but different surface hydrophobicities,they were not able to distinguish protein pairs with similar surface hydrophobicities. This corroborates that displacer-protein hydrophobic interactions play an important role for this class of selective displacers. Important functional group moieties were established and efficient displacers were identified. These results demonstrate that the design of chemically selective displacers requires a delicate balance between the abilities to displace proteins from the resin and to bind to a selected protein. The use of robotic screening of displacers will enable the extension of chemically selective displacement chromatography beyond hydrophobic displacer-protein interactions to other secondary interactions and more selective displacement systems.
机译：开发了一种机器人高通量置换剂筛，并用于在阳离子交换色谱法中鉴定几种蛋白质对的化学选择性置换剂。这种自动筛选功能可在较短的时间内评估各种实验条件。对这些蛋白质对在多种浓度下评估置换蛋白，并构建了DC-50图。还构建了选择性途径图，并建立了用于选择性和排他性分离的不同机制。重要的是，发现选择性置换可保留多个蛋白质对，这证明该技术可用于一系列蛋白质系统。尽管化学选择性置换剂能够分离在离子交换中具有相似保留但表面疏水性不同的蛋白质对，但它们无法区分具有相似表面疏水性的蛋白质对。这证实了置换蛋白与蛋白质的疏水相互作用在这类选择性置换剂中起着重要的作用。建立了重要的官能团部分，并确定了有效的置换剂。这些结果表明，化学选择性置换剂的设计要求在从树脂置换蛋白质和与选定蛋白质结合的能力之间达到微妙的平衡。机器人置换剂筛选的使用将使化学选择性置换色谱法从疏水性置换剂-蛋白质相互作用扩展到其他次级相互作用和更具选择性的置换系统。

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