Bioseparations


PROTEIN INTERACTIONS: PRECIPITATION AND CRYSTALLIZATION 

Protein-protein interactions are central to many biological and biotechnology processes. We are developing molecular-thermodynamic descriptions of protein phase equilibria in electrolyte solutions, providing a framework for the design and optimization of protein separation systems such as precipitation and crystallization. Salt-induced precipitation is extensively used in industry as a first purification step, and while crystallization provides the purest form of proteins, finding conditions for crystallization remains the limiting and least understood step in X-ray crystallographic determination of protein crystal structure. Protein aggregation results in the loss of biological activity, and misfolding and formation of insoluble deposits are observed in debilitating diseases (e.g., Alzheimer’s, Parkinson’s and Huntington’s diseases), in biotechnology (inclusion body formation) and in the pharmaceutical industry (protein formulation). We combine experimental methods and computer simulations to study protein interactions. 

As a first step in determining protein phase behavior, we consider proteins as spheres interacting via a two-body potential that depends on the physicochemical properties of the protein, the electrolyte and properties of the solution such as temperature and pH. Intermolecular interactions are measured using low angle laser light scattering, dynamic light scattering, membrane osmometry, fluorescence anisotropy and cloud point measurements. Chromatography also provides a source of data on specific protein-protein interactions. Integral equation approximations provide the link between potentials of mean force (pmf) and thermodynamic properties to yield the equilibrium phase behavior of the systems.


ELECTROPHORETIC SEPARATION OF DNA

Electrophoresis has been a longstanding, useful technique for separating DNA. It is the basis for many important techniques in molecular biology, including DNA restriction fragment mapping, DNA sequencing, Southern blotting, DNA fingerprinting and Dnase footprinting. It has typically been performed in slab gels, but the advent of capillary electrophoresis (CE) has expanded the use of DNA separations by reducing the time of separation. Typically, gel media have been used to effect a length-dependent separation in CE. In early work, we demonstrated that slow reptation-based DNA separations in gels can be replaced by the use of dilute polymer solutions as the separating agent in CE. We have shown that the mechanism of DNA separation is based a transient entanglement coupling of DNA with polymer, where the release time of the DNA is a function of DNA length, thereby providing length-dependent resolution. 

We have confirmed this new separation mechanism using epifluorescence video microscopy. By observing single-molecule DNA-polymer entanglements directly as DNA electrophoreses through a capillary, we see that DNA/polymer collisions depend on the size and concentration of each species, and that multiple entanglements are possible. We have obtained data on the entanglement time as a function of the Porod-Kratky persistence length of the polymer (a measure of its stiffness), the collision frequency and probability distribution of multiple entanglements. Using these data, a mechanistic model for DNA separations has been developed, and is able to predict DNA separations as a function of DNA size and polymer properties. 


© Harvey Blanch 2013