Polynucleotide High Performance Affinity Chromatography

描述（由申请人提供）：目的是提高转录因子的DNA亲和色谱性能，并增加对遗传调控的理解。为此，我们将： 1.开发我们称之为“合理捕获”的方案，以使寡核苷酸捕获能够针对特定蛋白质轻松优化。以前，我们已经开发了一种称为“寡核苷酸捕获”的方法，其中探针DNA以非常低的浓度与细胞提取物混合，并与各种竞争对手混合，从而减少非特异性结合。然后通过色谱法快速分离蛋白质-DNA复合物，并且转录因子以高纯度状态洗脱，通常是均质的。目前的方法需要很长时间来完善。我们将使用电泳迁移率变动分析来开发一种严格、合理的方法，以达到成功纯化的正确条件。 2.将合理捕获应用于三种非常不同的转录因子将显示它是否对不同的蛋白质起作用。然后将该方法应用于其他三种转录因子，以表明它对来自不同器官的不同蛋白质有效。 3.将开发一种称为启动子捕获的新方法。c-jun启动子只有200 bp左右。我们将扩展这种捕获的概念，以纯化整个转录前起始复合物（PIC），并通过蛋白质组学方法使用胰蛋白酶消化和质谱仪表征其组分蛋白。这一技术将能够识别事先知情同意的所有组成部分，包括目前未知的组成部分。该方法，沿着与合理的捕获，然后将被用来纯化三个转录因子结合该启动子，以研究转录调控细胞信号传导/磷酸化在HeLa细胞中响应于血清有丝分裂原。这些转录因子之一是目前未知的，将在这里首次表征。

DESCRIPTION (provided by applicant): The goal is to improve the performance DNA-affinity chromatography of transcription factors and to increase understanding of genetic regulation. To accomplish this we will: 1. Develop protocols we call "rational trapping" to allow oligonucleotide trapping to be easily optimized for a particular protein. Previously, we had developed a method called "oligonucleotide trapping" in which a probe DNA is mixed with cell extract at very low concentrations and with various competitors which lessen nonspecific binding. The protein-DNA complex is then rapidly isolated by chromatography and the transcription factor elutes in a high state of purity, typically homogeneous. The current method takes a long time to refine. We will use electrophoretic mobility shift assays to develop a rigorous, rational way to arrive at the correct conditions for successful purification. 2. The application of rational trapping to three very different transcription factors will show if it functions well for diverse proteins. The method will then be applied to three other transcription factors to show that it works well for diverse proteins from diverse organsims. 3. A new method, called promoter trapping, will be developed. The c-jun promoter is only about 200 bp. We will extend this trapping concept to purify the entire transcription pre-initiation complex (PIC) and characterize its component proteins by a proteomics approach using trypsin digestion and the mass spectrometers. This technique will allow identification of all components of the PIC, including ones which are currently unknown. The method, along with rational trapping, will then be used to purify three of the transcription factors bound by this promoter to investigate transcriptional regulation by cell signaling/phosphorylation in HeLa cells in response to serum mitogens. One of these transcription factors is currently unknown and will be characterized here for the first time.