Molecular Thermodynamics of Protein Interactions; Applications to Protein Separations

9530793蛋白质的白兰地回收和纯化在生物技术中至关重要。分离和纯化过程的设计和优化需要了解蛋白质在复杂水溶液中的行为，在复杂的水溶液中，盐、聚合物或其他溶质可能是稀释的或浓缩的。这项建议的目标是开发一种分离工艺。我们特别关注蛋白质的盐沉淀和蛋白质结晶；然而，蛋白质的溶液行为对于治疗性蛋白质的稳定性和配方以及了解分子间反应也很重要。这项建议是为了继续支持美国国家科学基金会的一个为期两年的项目：蛋白质在水溶液中沉淀的热力学。这种方法是在分子水平上用两体势的平均力(PMF)来描述蛋白质的溶液行为，PMF描述了两个蛋白质分子之间的整体相互作用。PMF由蛋白质的物理化学性质、溶液的性质和温度决定。从PMF可以获得热力学溶液性质来预测蛋白质沉淀的相平衡性质。计划利用液态积分方程式理论来发展蛋白质溶液的可压缩性和亥姆霍兹能量的新表达式；从这些表达式可以得到相平衡。这种建模方法与一个实验程序相结合，以确定一些模型蛋白质的相平衡，并特别注意从混合物中选择性地沉淀目标蛋白质。此外，还将对特定的蛋白质相互作用进行实验量化。低角度激光散射和膜渗透测量将提供有关分子间作用力和聚集的信息。特定的蛋白质-蛋白质相互作用将通过动态光散射进行检测。静电和疏水对PMF的贡献将通过系统地改变模型蛋白质上的氨基酸残基来探索。具体的离子-蛋白质相互作用将使用差示折射法、氯核磁共振和蛋白质滴定进行量化。*拟议研究的另一个组成部分是检查有利于蛋白质结晶的溶液条件。我们将通过测定一些蛋白质在结晶溶剂中的渗透第二维里系数来检验这一假设，即结晶溶剂导致蛋白质之间的弱吸引相互作用，而强吸引条件有利于无定形沉淀。它计划对一个由初步数据支持的想法进行批判性调查，即。当蛋白质的渗透第二维里系数略为负，但不是很大时，这种结晶最有可能发生。

9530793 Blanch Recovery and purification of proteins is of vital importance in biotechnology. Design and optimization of separation and purification processes requires an understanding of the behavior of proteins in complex aqueous solutions, where salts, polymers or other solutes may be dilute or concentrated. The objective of this proposal is to develop a separation processes. We are concerned in particular with protein precipitation by salts and with protein crystallization; however, protein solution behavior is also important in stability and formulation of therapeutic proteins, and in understanding intermolecular reactions. This proposal is for continued support of a two-year NSF project OThermodynamics of Protein Precipitation in Aqueous SolutionsO. The approach is to describe protein solution behavior on a molecular level, in terms of a two-body potential of mean force (PMF), which describes the overall interactions between two protein molecules. The PMF is determined by the physicochemical properties of the protein, the nature of the solution and temperature. From the PMF, thermodynamic solution properties may be obtained for predicting protein-precipitation phase-equilibrium properties. It is planned to employ liquid-state integral-equation theory to develop new expressions for the compressibility and Helmholtz energy of protein solutions; from these expressions phase equilibria are obtained. This modeling approach is coupled with an experimental program to determine the phase equilibria of a number of model proteins with particular attention given to selective precipitation of a target protein from a mixture. In addition, specific protein interactions will be experimentally quantified. Low-angle laser-light scattering and membrane osmometry will provide information on intermolecular forces and aggregation. Specific protein-protein interactions will be examined by dynamic light scattering. Electrostatic and hydrophobic contributions to the PMF will be probed by sy stematically changing amino-acid residues on the model proteins. Specific ion-protein interactions will be quantified using differential refractometry Cl NMR and protein titrations. *** A further component of the proposed research is to examine solution conditions which favor protein crystallization. The hypothesis that a crystallizing solvent results in weak attractive interactions between proteins, while strongly attractive conditions favor amorphous precipitation, will be examined by determining the osmotic second virial coefficients for a number of proteins in crystallizing solvents. It is planned to investigate critically an idea supported by preliminary data, viz. that crystallization is most likely when the osmotic second virial coefficient of a protein is slightly, but not strongly, negative.