title: Automated production of functional membrane proteins using eukaryotic cell-free translation systems.
abstract: Due to their high abundance and pharmacological relevance there is a growing demand for the efficient production of functional membrane proteins. In this context, cell-free protein synthesis represents a valuable alternative that allows for the high-throughput synthesis of functional membrane proteins. Here, we demonstrate the potential of our cell-free protein synthesis system, based on lysates from cultured Spodoptera frugiperda 21 cells, to produce pro- and eukaryotic membrane proteins with individual topological characteristics in an automated fashion. Analytical techniques, including confocal laser scanning microscopy, fluorescence detection of eYFP fusion proteins in a microplate reader and in-gel fluorescence of statistically incorporated fluorescent amino acid derivatives were employed. The reproducibility of our automated synthesis approach is underlined by coefficients of variation below 7.2%. Moreover, the functionality of the cell-free synthesized potassium channel KcsA was analyzed electrophysiologically. Finally, we expanded our cell-free membrane protein synthesis system by an orthogonal tRNA/synthetase pair for the site-directed incorporation of p-Azido-l-phenylalanine based on stop codon suppression. Incorporation was optimized by performance of a two-dimensional screening with different Mg(2+) and lysate concentrations. Subsequently, the selective modification of membrane proteins with incorporated p-Azido-l-phenylalanine was exemplified by Staudinger ligation with a phosphine-based fluorescence dye.
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, 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, 14476 Potsdam, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Kortt
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, 14476 Potsdam, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Henkel
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, 14476 Potsdam, Germany.
authors.forename: Srujan K
authors.initials: SK
authors.lastname: Dondapati
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, 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, 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, 14476 Potsdam, Germany. Electronic address: stefan.kubick@izi-bb.fraunhofer.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Kubick
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry; D000818:Animals; D051398:Aquaporin 1 / Q000737:chemistry*; D001386:Azides / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D001436:Bacteriorhodopsins / Q000737:chemistry; D066246:ErbB Receptors / Q000737:chemistry*; D066257:Heparin-binding EGF-like Growth Factor / Q000737:chemistry*; D008164:Luminescent Proteins / Q000737:chemistry; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D010957:Plasmids; D015221:Potassium Channels / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D001386:Azides; D001426:Bacterial Proteins; C587885:Hbegf protein, rat; D066257:Heparin-binding EGF-like Growth Factor; D008164:Luminescent Proteins; D015221:Potassium Channels; D011993:Recombinant Fusion Proteins; C107790:prokaryotic potassium channel; C054562:yellow fluorescent protein, Bacteria; D051398:Aquaporin 1; C027162:4-azidophenylalanine; D010649:Phenylalanine; D001436:Bacteriorhodopsins; C512478:EGFR protein, human; D066246:ErbB Receptors; D000604:Amino Acyl-tRNA Synthetases
keywords: Automation; Electrophysiology; Eukaryotic cell-free protein synthesis; Fluorescence modification; Integral membrane protein; Non-canonical amino acid