Polynucleotide High Performance Affinity Chromatography

描述(申请人提供)：目标是改进转录因子的DNA亲和层析的性能，并增加对遗传调节的理解。要做到这一点，我们将： 1.开发我们称为“合理捕获”的协议，使寡核苷酸捕获能够针对特定蛋白质轻松优化。在此之前，我们开发了一种称为“寡核苷酸捕获”的方法，在这种方法中，探针DNA在非常低的浓度下与细胞提取液混合，并与各种竞争对手混合，从而减少非特异性结合。然后通过层析快速分离蛋白质-DNA复合体，转录因子在高纯度状态下洗脱，通常是均一状态。目前的方法需要很长时间才能完善。我们将使用电泳迁移率变化分析来开发一种严格、合理的方法来获得成功纯化的正确条件。 2.对三种截然不同的转录因子进行合理捕获，可以发现它对不同的蛋白质是否有很好的作用。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起到了很好的作用。 3.将开发一种新的方法，称为启动子捕获。C-jun启动子只有200个碱基左右。我们将把这一捕获概念扩展到纯化整个转录前起始复合体(PIC)，并通过使用胰酶消化和质谱仪的蛋白质组学方法来表征其组成蛋白。这项技术将允许识别PIC的所有组件，包括目前未知的组件。该方法与合理的捕获方法一起，将被用于纯化该启动子结合的三个转录因子，以研究细胞信号/磷酸化对血清有丝分裂原反应的HeLa细胞的转录调控。这些转录因子中的一个目前尚不清楚，将首次在这里进行鉴定。

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.