Protein Arrays: Phosphorylation-dependent Interactions

蛋白质阵列：磷酸化依赖性相互作用

• 批准号: 6403812
• 负责人: PAZHANI SUNDARAM
• 金额: $ 12.24万
• 依托单位: RECOMBINANT TECHNOLOGIES, LLC
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6403812
• 关键词: Escherichia coli; affinity chromatography; biological signal transduction; high throughput technology; method development; phosphopeptides; phosphorylation; protein protein interaction; protein structure function; protein tyrosine kinase; thiols

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.

描述（由申请人提供）：蛋白质-蛋白质相互作用是核心 生物系统中的信号转导过程。跨膜 受体酪氨酸激酶（RTK）在细胞的许多方面发挥着重要作用 成长和行为。激活的 RTK，包括经过充分研究的 RTK 之一 PDGF-β受体，已被证明可以结合十多个信号 含有 Sre 同源 2 (SH2) 结构域的转导分子。这些 相互作用取决于特定的磷酸酪氨酸残基的识别 各个 SH2 域的序列上下文。在拟议的研究中， 选定的重组 SH2 结构域构建体将在大肠杆菌中表达，并且 通过亲和层析纯化。在衍生玻璃上测试蛋白质阵列 幻灯片将使用 PI3 激酶的 SH2 结构域进行优化，基于其 与合成磷酸肽结合。额外的重组 SH2 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 PDGF 受体。我们预测该方法将 允许快速读出 PDG 上哪些特定酪氨酸被磷酸化 受体。这些研究旨在优化阵列和结合参数， 从而开发一种高通量检测磷酸化的新策略 依赖性蛋白质：蛋白质相互作用。 拟议的商业应用： 将开发商业 SH2 域阵列以帮助高通量筛选 涉及细胞信号转导的新相互作用和/或可能的新激酶。 承包 SH2 蛋白交易的高通量筛选项目。