Protein Arrays: Phosphorylation-dependent Interactions

描述（由申请人提供）：蛋白质-蛋白质相互作用是核心 生物系统中的信号传导过程。跨膜 受体酪氨酸激酶（receptor tyrosine kinase，RTKs）在细胞的许多方面发挥重要作用， 成长和行为。激活的RTK，包括一个研究得很好的 PDGF-β受体，已被证明可以结合十几种信号 在一些实施方案中，所述转导分子包含Sre同源2（SH 2）结构域。这些 相互作用依赖于识别磷酸酪氨酸残基在特定的 序列上下文的各种SH 2结构域。在拟议的研究中， 选择的重组SH 2结构域构建体将在E.杆菌和 通过亲和层析纯化。在衍生玻璃上测试蛋白质阵列 将根据其特性，使用PI 3-激酶的SH 2结构域优化载玻片。 与合成磷酸肽结合。另外的重组SH 2结构域将 排列并允许与表达V-sis的细胞提取物相互作用， 激酶死亡或野生型血小板源生长因子受体。我们预测，这种方法将 允许快速读出PDG上哪些特定酪氨酸被磷酸化 受体的这些研究旨在优化阵列和结合参数， 从而为磷酸化的高通量检测提供了一种新的策略 依赖蛋白质：蛋白质相互作用。 拟定商业应用： 将开发商业SH 2结构域阵列以帮助高通量筛选 涉及细胞信号转导的新型相互作用和/或可能的新型激酶。 承接SH 2蛋白高通量筛选项目。

DESCRIPTION (provided by applicant): Protein-protein interactions are central to the process of signal transduction in biological systems. Transmembrane receptor tyrosine kinase (RTKs) play important roles in many aspects of cell growth and behavior. Activated RTKs, including one of the well-studied PDGF-beta receptor, have been shown to bind more than a dozen signal transduction molecules that contain Sre homology 2 (SH2) domains. These interactions depend on the recognition of phosphotyrosine residues in specific sequence contexts by the various SH2 domains. In the proposed research, selected recombinant SH2 domain constructs will be expressed in E. coli and purified by affinity chromatography. Test protein arrays on derivatized glass slides will be optimized with an SH2 domain from PI3-kinase based on its binding to a synthetic phosphopeptide. Additional recombinant SH2 domains will be arrayed and allowed to interact with extracts of cells expressing V-sis with a kinase dead or wild type PDGF receptor. We predict that this method will allow a rapid readout of which specific tyrosines are phosphorylated on the PDG receptor. These studies will aim to optimize array and binding parameters and thus to develop a new strategy for high throughput detection of phosphorylation dependent protein: protein interactions. PROPOSED COMMERCIAL APPLICATION: Commercial SH2 domain arrays will be developed to aid high throughput screening of novel interactions involved in cellular signal transduction and or possible novel kinases. Contracting in projects for high throughput screening of SH2 protein transactions.