title: High-yield cell-free synthesis of human EGFR by IRES-mediated protein translation in a continuous exchange cell-free reaction format.
abstract: Cell-free protein synthesis systems derived from eukaryotic sources often provide comparatively low amounts of several μg per ml of de novo synthesized membrane protein. In order to overcome this, we herein demonstrate the high-yield cell-free synthesis of the human EGFR in a microsome-containing system derived from cultured Sf21 cells. Yields were increased more than 100-fold to more than 285 μg/ml by combination of IRES-mediated protein translation with a continuous exchange cell-free reaction format that allowed for prolonged reaction lifetimes exceeding 24 hours. In addition, an orthogonal cell-free translation system is presented that enabled the site-directed incorporation of p-Azido-L-phenylalanine by amber suppression. Functionality of cell-free synthesized receptor molecules is demonstrated by investigation of autophosphorylation activity in the absence of ligand and interaction with the cell-free synthesized adapter molecule Grb2.
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, D-14476 Potsdam, Germany.
authors.forename: Robert B
authors.initials: RB
authors.lastname: Quast
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, D-14476 Potsdam, Germany.
authors.forename: Andrei
authors.initials: A
authors.lastname: Sonnabend
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, D-14476 Potsdam, Germany.
authors.forename: Marlitt
authors.initials: M
authors.lastname: Stech
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, D-14476 Potsdam, Germany.
authors.forename: Doreen A
authors.initials: DA
authors.lastname: Wüstenhagen
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, D-14476 Potsdam, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Kubick
mesh_terms: D000818:Animals; D002474:Cell-Free System; D066246:ErbB Receptors / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D000067556:Internal Ribosome Entry Sites; D008861:Microsomes / Q000378:metabolism*; D009154:Mutation; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D000067556:Internal Ribosome Entry Sites; C512478:EGFR protein, human; D066246:ErbB Receptors