Transcription factors and other DNA binding proteins must be characterized to understand genetic regulation, DNA repair, and the life cycle of the cell -- all important goals for the improvement of human health and well-being. Affinity chromatography provides the only rational way of purifying many of these components. Late in the last project period we made three important new discoveries which will be characterized over the next budget period. Here, we improve this chromatography by: 1. Develop CNBr-activated Silica into a usable RPLC support. We recently adapted the cyanogen bromide activation chemistry to silica. This new approach should result in efficient coupling with few chromatographic artifacts 2. Catalytic chromatography will be tested by the resolution of DNA polymerases to answer important questions about the advantages of this chromatographic mode. We recently showed that affinity chromatography using both binding- and catalyticspecificity, i.e., """"""""catalytic chromatography,"""""""" gives superior yield and purity of a restriction enzyme (16). We will learn if catalytic chromatography can perform separations impossible by other means as we suspect, relying on subtle differences in catalytic mechanism. This approach may also selectively purify active enzyme away from inactive forms. 3. A model of catalytic chromatography will be constructed relating retention time, binding affinity, catalytic rate, and protein load. 4. Characterize those conditions that give highest purity and yield with a new """"""""trapping"""""""" protocol affinity chromatography. We have found ways in which very low (nM) concentrations of DNA can be used to """"""""trap"""""""" a particular transcription factor from the complex mixture of DNA-binding proteins in cells. This method has already been shown to give higher purity than other procedures. This new method will be improve by defining those conditions that work best. 5. CNBr-silica and the catalytic and trapping chromatography modes will be combined to test the resolution and other properties of these hybrid technologies. These five years of study will make affinity chromatography less empirical and more reliable, improving progress across a broad cross-section of the biomedical sciences.
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