The role of protein-protein interactions in the phase behavior of aqueous protein solutions

Purification of recombinant proteins is of vital importance to the biotechnological and pharmaceutical industries. Salt-induced precipitation and crystallization are commonly used techniques for protein purification. Recombinant proteins are generated in aqueous solutions where the separation is complicated by the presence of contaminants such as polymers, salts, and other proteins. Optimization of separation processes requires an understanding of the intermolecular forces between various solution components. The goal of this research is to develop a simple molecular-thermodynamic description of protein-protein interactions and protein phase behavior in aqueous solutions containing water, electrolytes, and proteins.; In this work, we focus on the phase separation of proteins induced by addition of salt. Our molecular-thermodynamic model is based on a two-body potential of mean force (PMF) which is a measure of the net interaction between two proteins. Many factors influence the strength of this interaction; these include the physicochemical properties of the protein, the nature of the solvent, the type and concentration of salt, the presence of other proteins, pH, and temperature. The DLVO (Derjaguin-Landau-Verwey-Overbeek) potential of mean force incorporates interactions due to protein size, electric double-layer interactions, and dispersion attraction. Interactions due to other (non-DLVO) attraction are here incorporated through a specific-interaction potential. Parameters for the attractive specific-interaction potential are regressed from experimental data (osmotic-pressure measurements and fluorescence-anisotropy measurements) obtained in dilute aqueous, saline protein solutions.; We measure interactions for hen egg-white lysozyme, ovalbumin, bovine serum albumin, and for a peptide derived from T4 lysozyme. The magnitude of the regressed parameters indicates that the primary effect of high salt concentrations is to enhance hydrophobic forces between protein molecules. Also, hydrogen-bond formation in the peptide solutions is enhanced by the presence of salt and addition of a co-solvent, trifluoroethanol. The strength of attractive forces between the peptide molecules depends on the degree of secondary structure as determined from circular-dichroism measurements.; The thermodynamic properties and phase behavior of proteins are described by a statistical-mechanical model that combines the Random Phase Approximation and the Statistical Associating Fluid Theory. Using this model, expressions are derived for the chemical potential of the protein and for the equation of state of the protein solution. Protein precipitation is modeled as a liquid-liquid equilibrium. The distribution coefficient characterizes phase separation; it is calculated from the protein concentrations in these two liquid phases. Phase equilibria are calculated for two globular proteins, hen egg-white lysozyme and ovalbumin, in both single-protein solutions and two-protein solutions. Semi-quantitative agreement with experimental precipitation data is achieved. The molecular theory derived here is useful for describing macromolecular phase-equilibria in protein solutions. These equilibria are of interest in industrial biotechnology and also in medicine where serious neurological diseases (e.g., Alzheimer's) involve protein aggregation and deposition.