Reconstitution of tyrosine kinase signaling in cell-free systems: Synthetic membrane protein dimerization and lipid modification

该项目的主要目标是：- 组成型活性EGF受体的体外产生和化学修饰（通过用于永久二聚化受体的遗传编码的非天然氨基酸）及其下游效应物RhoA（通过引入由非天然氨基酸促进的脂质部分）-通过化学方法和无细胞系统中天然存在的翻译后修饰的组合在脂质双层中重构整合膜蛋白和膜相关蛋白（通过遗传编码的非天然氨基酸和新的异双功能接头化学）-用于蛋白质二聚化的新的异双功能接头化学的开发-新的tRNA/在各种体外翻译系统中将非天然氨基酸掺入蛋白质的tRNA合成酶对-通过磷酸化检测监测体外合成和组成活性膜蛋白的功能（基于抗体的测定）和蛋白质-蛋白质相互作用分析（质谱分析）-鉴定与EGF受体下游信号通路相关的新型抑制成分

The Main Goals of the project are:- Reconstitution of EGFR depended signaling pathways in cell-free systems- In vitro generation and chemical modification of constitutively active EGF-Receptor (via genetically encoded unnatural amino acids utilized to dimerize the receptor permanently) and its downstream effector RhoA (via the introduction of lipid moieties facilitated by unnatural amino acids)- Reconstitution of integral membrane proteins and membrane associated proteins in lipid bilayers by combination of chemical methods and naturally occurring posttranslational modifications in cell-free systems (via genetically encoded unnatural amino acids and novel heterobisfunctional linker chemistries)- Development of novel heterobisfunctional linker chemistries for protein dimerization- Implementation of novel tRNA/tRNA synthetase pairs for the incorporation of unnatural amino acids into proteins in various in vitro translation systems- Monitoring functionality of in vitro synthesized and constitutive active membrane proteins through detection of phosphorylation (Antibody based Assays) and Protein-Protein interaction analysis (mass spectrometry Assays)- Identification of novel inhibitory components with regard to the EGF-receptor’s downstream signaling pathway