Polynucleotide High Performance Affinity Chromatography

描述(申请人提供)：转录因子和其他DNA 必须对结合蛋白进行表征才能理解遗传调控，DNA 修复，以及细胞的生命周期--这些都是 增进人类健康福祉。亲和层析提供 提纯这些成分的唯一合理方法。最后一天晚些时候 在项目期间，我们有了三个重要的新发现，它们将是 下一个预算期的特点。在这里，我们改进了这种层析 通过：1.将溴化碳活化的二氧化硅发展成可用的RPLC载体。我们最近 将溴化氰活化化学改编为二氧化硅。这一新方法 应能与较少的色谱柱2进行有效的偶联。 催化层析将通过将DNA聚合酶分解为 回答有关这种层析模式优势的重要问题。 我们最近发现，亲和层析同时使用结合和 催化专一性，即“催化层析”，提供了更高的产率。 和限制性内切酶(16)的纯度。我们将了解是否具有催化作用 正如我们怀疑的那样，层析可以用其他方法进行不可能的分离， 依靠催化机理上的细微差别。这种方法还可以 选择性地从非活性形式中提纯活性酶。3.一种模式 将构建与保留时间、结合 亲和力、催化速率和蛋白质负载量。4.描述这些条件 提供最高纯度和产量，并具有新的“陷阱”协议亲和力 层析法。我们已经找到了极低浓度(NM)DNA的方法 可以用来从复合体中“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是 提供比其他程序更高的纯度。这一新方法将被改进为 定义最有效的条件。5.溴化碳-二氧化硅及其催化剂和 将结合捕获层析模式来测试分辨率和其他 这些混合技术的特性。这五年的学习将使 亲和层析更少经验和更可靠，正在改进进展 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.