POLYNUCLEOTIDE HIGH PERFORMANCE AFFINITY CHROMATOGRAPHY

描述：转录因子的转录调控是 S的基因表达与细胞生物学。这些蛋白质的纯化方法是 是经验主义的。已知的转录因子有数百种，但为数不多 都有很好的特点。可以使用的最强大的技术之一 用DNA亲和层析法对这些蛋白质进行纯化。这个 拟议的工作将开发一种合理的转录因子方法 采用高效DNA亲和层析法进行纯化。 这项建议的具体目标是： 1.要发现哪种层析载体在 研究实验室。(将考虑三种不同的支持： 琼脂糖凝胶、HEMA和硅胶。琼脂糖将被用作历史 这是标准的，因为以前的大多数工作都是在Sepharose支持下完成的。这个 另外两种是更现代化的高效支架。) 2.确定将DNA连接到载体上的首选方法。 (有许多化学方法可以将DNA连接到载体上。 根据附着部位的不同，化学物质有时意味着 使附着的DNA的一部分失活以与DNA结合相互作用 蛋白质。用酶将DNA连接到载体上是一种方法 由调查人员开发的。对于一些与DNA结合的蛋白质，他们发现 这将是首选的方法。然而，附加的复杂性 通常情况下，当酶结合方法时，DNA含量更大 使用的是。因此，DNA和污染物之间的非特异性相互作用 来自细胞的蛋白质可能是酶附着的问题。) 3.确定具有最佳性能的DNA序列。(在那里 有各种报告表明，短片段的DNA对人的健康更好 DNA蛋白质的纯化。然而，其他报告表明，长时间 识别序列的重复是最佳的。) 4.优化色谱柱洗脱条件。一系列变量 已经被用来从DNA亲和柱上洗脱DNA结合蛋白。 包括改变盐浓度，改变温度， 加入有机溶剂，加入金属离子，并加入竞争DNA。 5.将这些技术分别应用于三种不同类别的DNA 结合蛋白。所有的初始优化工作都将涉及 紫胶阻遏蛋白的纯化。这种蛋白质是研究最多的 转录调节因子。它有螺旋-旋转-螺旋，C/EBP蛋白有 亮氨酸拉链基序和TFIIIA基序具有锌指基序。后 另外两种蛋白--紫胶蛋白条件的优化 都将经过同样严格的优化。 6.最后，从中获得的理解将被用来净化 B3激活蛋白。

DESCRIPTION: Transcriptional regulation by transcription factors is key to gene expression and the cell s biology. Purification of these proteins is approached empirically. Hundreds of transcription factors are known but few are well characterized. One of the most powerful techniques available for the purification of these proteins is DNA-affinity chromatography. The proposed work will develop a rational approach to transcription factor purifications using high performance DNA-affinity chromatography. The specific aims of this proposal are: 1.To discover which chromatographic supports provide the best performance in the research laboratory. (Three different supports will be considered: Sepharose, HEMA and silica. The Sepharose will be used as the historical standard since most previous work was done with a Sepharose support. The other two are more modern high efficiency supports.) 2. To determine the preferred method for attaching DNA to a support. (There are numerous chemical means of attaching DNA to the supports. Depending on the site of attachment, the chemical means sometimes inactivates a part of the attached DNA for interaction with the DNA binding proteins. Enzymatic attachment of the DNA to the support is a method developed by the investigators. For some DNA-bind proteins, they have found this to be the preferred method. However, the complexity of the attached DNA is typically by necessity greater when the enzymatic attachment method is used. Therefore nonspecific interactions between the DNA and contaminant proteins from the cells can be a problem with enzymatic attachment.) 3. To determine the DNA sequence which gives optimal performance. (There are various reports that suggest that short pieces of DNA are better for the purification of DNA proteins. However, other reports indicate that long repeats of recognition sequences are optimal.) 4. To optimize the elution protocol for the column. A range of variables have been used to elute DNA-binding proteins from DNA-affinity columns. These include changing the salt concentration, changing the temperature, adding an organic solvent, adding metal ions , and adding a competing DNA. 5. To apply each of these techniques to three different classes of DNA binding proteins. All of the initial optimization work will involve the purification of the lac repressor protein. This protein is the most studied transcription regulator. It has the helix-turn-helix, the C/EBP protein has the leucine zipper motif and the TFIIIA has the Zn-finger motif. After the optimization of the conditions for the lac proteins, the other two proteins will be taken through the same sort of rigorous optimization. 6. Finally, the understanding gained from this will be used to purify the B3 activator protein.