Cell-Free Expression and Purification of the Fragments of the Receptor Tyrosine Kinases of the EGFR Family, Containing the Transmembrane Domain with the Juxtamembrane Region, for Structural Studies

摘要

The EGFR/HER receptor family of an epidermal growth factor represents an important class of the receptor tyrosine kinases playing the key role in the control of cell growth and differentiation in mammalian cells, as well as in the development of a number of pathological processes, including oncogenesis. Binding of a ligand to the extracellular domains initiates switching of the EGFR/HER receptor between the alternative dimeric states that causes the allosteric activation of kinase domains in cell cytoplasm. The transmembrane (TM) domain and adjacent flexible regions alternatively interacting with either membrane surface or kinase domains are directly involved in the complex conformational transition in EGFR/HERs. Here we report on a highly efficient system of the cell free production of the EGFR/HER TM domains with functionally important juxtamembrane (JM) regions for the investigation of the molecular basis of biochemical signal transduction across the cell membrane. To increase the efficiency of synthesis of the EGFR/HER TM-JM fragments of the receptors, we used two N-terminal expression tags, which significantly increased the protein yield. In the case of the TM-JM fragments of EGFR (residues 638–692) and HER2 (residues 644–700), the method allowed us to obtain milligram quantities of the ~(13)C,~(15)N-labeled protein for structural and biophysical investigations in the membrane-mimicking environments using high-resolution heteronuclear NMR spectroscopy.