Molecular Thermodynamics of Protein Interactions; Applications to Protein Separations

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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.