Fan W, Bakir U, Glatz C E
Department of Chemical Engineering, Iowa State University, 2114 Sweeney Hall, Ames, Iowa 50011-2230, USA.
Biotechnol Bioeng. 1998 Aug 20;59(4):461-70. doi: 10.1002/(sici)1097-0290(19980820)59:4<461::aid-bit9>3.0.co;2-e.
Protein partitioning in aqueous two-phase systems based on phase-forming polymers is strongly affected by the net charge of the protein, but a thermodynamic description of the charge effects has been hindered by conflicting results. Many of the difficulties could be because of problems in isolating electrochemical effects from other interactions of phase components. We explored charge effects on protein partitioning in poly(ethylene glycol)-dextran two-phase systems by using two series of genetically engineered charge modifications of bacteriophage T4 lysozyme produced in Escherichia coli. The two series, one in the form of charged-fusion tails and the other in the form of charge-change point mutations, provided matching net charges but very different polarity. Partition coefficients of both series were obtained and interfacial potential differences of the phase systems were measured. Multi-angle laser light scattering measurements were also performed to determine second virial coefficients. A semi-empirical model accounting for the roles of both charge and non-charge effects on protein partitioning behavior is proposed, and the results predicted from the model are compared to the results from the experiments.
基于成相聚合物的双水相体系中蛋白质的分配受到蛋白质净电荷的强烈影响，但电荷效应的热力学描述一直受到相互矛盾的结果的阻碍。许多困难可能是由于难以将电化学效应与相组分的其他相互作用分离开来。我们通过使用在大肠杆菌中产生的两系列噬菌体T4溶菌酶的基因工程电荷修饰，探索了聚乙二醇-葡聚糖双水相体系中电荷对蛋白质分配的影响。这两个系列，一个是带电荷融合尾的形式，另一个是电荷变化点突变的形式，提供了匹配的净电荷但极性非常不同。获得了两个系列的分配系数，并测量了相体系的界面电位差。还进行了多角度激光光散射测量以确定第二维里系数。提出了一个半经验模型，该模型考虑了电荷和非电荷效应在蛋白质分配行为中的作用，并将模型预测的结果与实验结果进行了比较。
