Molecular Thermodynamics of Protein Interactions; Applications to Protein Separations

蛋白质的回收和纯化在生物技术中是至关重要的。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，其中盐，聚合物或其他溶质可能被稀释或浓缩。本建议的目标是开发一个分离过程。我们特别关注盐沉淀蛋白质和蛋白质结晶；然而，蛋白质溶液行为在治疗蛋白的稳定性和配方以及理解分子间反应中也很重要。该提案是为了继续支持一项为期两年的NSF项目：水溶液中蛋白质沉淀的热力学。该方法是在分子水平上描述蛋白质溶液的行为，根据两体平均力势（PMF），它描述了两个蛋白质分子之间的总体相互作用。PMF由蛋白质的物理化学性质、溶液的性质和温度决定。从PMF中，可以得到预测蛋白质沉淀相平衡性质的热力学溶液性质。计划运用液相积分方程理论，建立蛋白质溶液的可压缩性和亥姆霍兹能的新表达式；由这些表达式得到相平衡。这种建模方法与实验程序相结合，以确定一些模型蛋白质的相平衡，特别注意从混合物中选择性沉淀目标蛋白质。此外，具体的蛋白质相互作用将实验量化。低角度激光散射和膜渗透法将提供分子间作用力和聚集的信息。特定的蛋白质与蛋白质之间的相互作用将通过动态光散射来研究。静电和疏水对PMF的贡献将通过系统地改变模型蛋白上的氨基酸残基来探测。特定的离子-蛋白质相互作用将被定量使用差示折射法Cl核磁共振和蛋白质滴定。***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。结晶溶剂导致蛋白质之间弱吸引相互作用，而强吸引条件有利于无定形沉淀的假设，将通过确定结晶溶剂中许多蛋白质的渗透第二维里系数来检验。计划对初步数据所支持的一个观点进行批判性的研究，即当蛋白质的渗透第二维里系数为轻微而不是强烈的负时，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.