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

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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