Polynucleotide High Performance Affinity Chromatography

描述（申请人提供）：转录因子和其他DNA 结合蛋白必须被表征以理解遗传调节，DNA 修复，以及细胞的生命周期--这些都是研究的重要目标。 改善人类健康和福祉。亲和层析提供了 唯一合理的方法来提纯这些成分在最后一个 在项目期间，我们有三个重要的新发现， 在下一个预算期间。在这里，我们改进了这种色谱法 通过：1.将CNBr活化的二氧化硅开发成可用的RPLC载体。我们最近 使溴化氰活化化学适应二氧化硅。这种新方法 应该导致有效的偶联和很少的色谱伪像2。 催化色谱法将通过DNA聚合酶的分离度进行测试， 回答有关这种色谱模式优点的重要问题。 我们最近表明，亲和层析使用结合和 催化特异性，即，“催化色谱法，”给出了上级产率 和限制酶的纯度（16）。我们将了解到催化剂 色谱法可以进行我们所怀疑的通过其他手段不可能进行的分离， 依靠催化机制的细微差别。此方法还可 选择性地将活性酶从非活性形式中纯化出来。3.的典范 将构建与保留时间、结合 亲和力、催化速率和蛋白质载量。4.描述这些条件 其以新“捕获”方案亲和性得到最高纯度和产率 层析我们已经发现了非常低浓度（nM）的DNA 可用于从复合物中“捕获”特定的转录因子 细胞中DNA结合蛋白的混合物。这种方法已经被证明 得到比其他方法更高的纯度。这种新方法将通过以下措施加以改进： 确定最佳工作条件。5. CNBr-二氧化硅和催化剂， 将结合捕获色谱模式来测试分离度和其他 这些混合技术的特点。这五年的学习将使 亲和层析经验性少，更可靠，不断改进 横跨生物医学科学的广泛领域。

DESCRIPTION (provided by applicant): 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.