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

无细胞系统中酪氨酸激酶信号的重建：合成膜蛋白二聚化和脂质修饰

基本信息

批准号：
223401531
负责人：
Dr. Stefan Kubick
金额：
--
依托单位：
Institut für Pharmazeutische Technologie
依托单位国家：
德国
项目类别：
Priority Programmes
财政年份：
2012
资助国家：
德国
起止时间：
2011-12-31 至 2016-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/223401531?language=en
关键词：
Reconstitution tyrosine kinase signaling cell

项目摘要

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

该项目的主要目标是：-在无细胞系统中重建EGFR依赖的信号通路-体外产生和化学修饰具有结构性活性的EGF-受体(通过用于永久二聚化受体的遗传编码的非天然氨基酸)及其下游效应分子RhoA(通过引入由非天然氨基酸促进的脂类部分)-通过结合化学方法和无细胞系统中自然发生的翻译后修饰(通过遗传编码的非天然氨基酸和新的异双功能连接物化学)-开发用于蛋白质二聚的新型异双功能连接物化学不同体外翻译系统中非天然氨基酸加入蛋白质的tRNA/tRNA合成酶对-通过检测磷酸化(基于抗体的分析)和蛋白质-蛋白质相互作用分析(质谱分析)监测体外合成和构成的膜蛋白的功能-关于EGF受体下游信号通路的新抑制成分的鉴定