Evaluation of chemically selective displacer analogues for protein purification.
Evaluation of chemically selective displacer analogues for protein purification.
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DOI：
10.1021/ac900710f
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发表时间：
2009-07
影响因子：
7.4
通讯作者：
C. J. Morrison;C. Breneman;J. Moore;S. Cramer
中科院分区：
文献类型：
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作者：
C. J. Morrison;C. Breneman;J. Moore;S. Cramer
选择性置换剂N '1'-（4-甲基喹啉-2-基）乙烷-1，2-二胺二硝酸酯的分子类似物库被用来研究置换剂化学变化对其选择性分离效率的影响。使用机器人液体处理系统进行高通量筛选，以检查这些化合物在分批吸附系统中选择性置换蛋白质的能力。使用模型蛋白质对核糖核酸酶A/α-胰凝乳蛋白酶原A和细胞色素C/溶菌酶在强阳离子交换剂上进行实验。从模拟筛选数据构建选择性途径和DC-50图，结果表明置换剂分子设计的微小变化可对分离行为产生显著影响。具体而言，发现树脂和蛋白质相互作用部分的电荷密度和间距是重要的。筛选还鉴定了一种新的置换剂，4-甲基-2-哌嗪-1-基-喹啉，其产生比先前报道的原始化合物更选择性的置换。构建了空间质量作用动态亲和力图，以验证这种新的置换剂是作为化学选择性的，而不是空间质量作用选择性置换剂。最后，进行饱和转移差核磁共振实验来检查蛋白质-置换剂与这些置换剂和蛋白质对的相互作用。这些结果表明，如何微妙的变化，置换剂的设计可以用来微调的化学选择性置换剂的分离性能。
A library of molecular analogues to the selective displacer, N'1'-(4-methylquinolin-2-yl)ethane-1,2-diamine dinitrate, was employed to study the effects of changes in displacer chemistry on their efficacy for selective separations. High throughput screens were carried out using a robotic liquid handling system to examine the ability of these compounds to selectively displace proteins in batch adsorption systems. Experiments were conducted using the model protein pairs ribonuclease A/alpha-chymotrypsinogen A and cytochrome C/lysozyme on a strong cation exchanger. Selectivity pathway and DC-50 plots were constructed from the analogue screen data, and results indicated that minor changes in the molecular design of the displacer can have a significant impact on the separation behavior. Specifically, charge density and spacing of resin and protein interaction moieties were found to be important. The screen also identified a new displacer, 4-methyl-2-piperazin-1-yl-quinoline, which produced a more selective displacement than previously reported with the original compound. A steric mass action dynamic affinity plot was constructed to validate that this new displacer was acting as a chemically selective, rather than a steric mass action selective displacer. Finally, saturation transfer difference NMR experiments were conducted to examine protein-displacer interactions with these displacers and protein pairs. These results demonstrate how subtle changes in displacer design can be employed to fine-tune the separation performance of chemically selective displacers.