POLYNUCLEOTIDE HIGH PERFORMANCE AFFINITY CHROMATOGRAPHY

描述：转录因子的转录调控是 基因表达和细胞生物学。 这些蛋白质的纯化是 接近经验。 已知的转录因子有数百种， 被很好地表征。 最强大的技术之一， 这些蛋白质的纯化是DNA亲和层析。 的 建议的工作将发展一个合理的方法，转录因子 使用高效DNA亲和层析纯化。 这项建议的具体目标是： 1.发现哪些色谱支持物在以下方面提供最佳性能： 研究实验室。 （将考虑三种不同的支持： 琼脂糖凝胶、HEMA和二氧化硅。 琼脂糖凝胶将被用作历史样品。 标准，因为大多数以前的工作是在琼脂糖支持下进行的。 的 另外两个是更现代化的高效支架。 2. 确定将DNA连接到支持物上的首选方法。 （有许多化学方法可以将DNA连接到支持物上。 根据附着的部位，化学手段有时 使附着的DNA的一部分失活以与DNA结合物相互作用， proteins. DNA与载体的酶促连接是一种方法， 由调查人员开发。 对于一些DNA结合蛋白，他们发现 这是优选的方法。 然而，所附的复杂性 当酶连接方法 采用了 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. 以确定提供最佳性能的DNA序列。 （在 有各种各样的报告表明，短片段的DNA更适合 纯化DNA蛋白。 然而，其他报告表明，长期以来， 识别序列的重复是最佳的）。 4. 优化色谱柱的洗脱方案。 一系列变量 已被用于从DNA亲和柱上除去DNA结合蛋白。 这些包括改变盐的浓度，改变温度， 加入有机溶剂，加入金属离子，加入竞争DNA。 5. 将这些技术应用于三种不同类型的DNA 结合蛋白 所有初始优化工作都将涉及 纯化lac阻遏蛋白。 这种蛋白质是研究最多的 转录调节子 它有螺旋-转角-螺旋，C/EBP蛋白有 亮氨酸拉链基序和TFIIIA具有锌指基序。 后 对lac蛋白、其他两种蛋白的条件进行优化， 都将经过同样严格的优化。 6. 最后，从中获得的理解将用于净化 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.