title: Membrane assembly of the functional KcsA potassium channel in a vesicle-based eukaryotic cell-free translation system.
abstract: The potassium channel KcsA was heterologously expressed in a eukaryotic cell-free system. Both, the expression yields and functional analysis of the protein were reported. Qualitative and quantitative analyses of KcsA expression were performed by using (14)C-labeled leucine as one of the amino acids supplemented in the cell-free reaction mixture. There was a time dependent increase in the protein yield as well as the intensity of the native tetramer band in insect cell derived microsomes. Electrophysiology measurements demonstrated the functional activity of the microsomes harboring KcsA showing single-channel currents with the typical biophysical characteristics of the ion channel. The channel behavior was asymmetric and showed positive rectification with larger currents towards positive voltages. KcsA channel currents were effectively blocked by potassium selective barium (Ba(2+)). This functional demonstration of an ion channel in eukaryotic cell-free system has a large potential for future applications including drug screening, diagnostic applications and functional assessment of complex membrane proteins like GPCRs by coupling them to ion channels in cell-free systems. Furthermore, membrane proteins can be expressed directly from linear DNA templates within 90 min, eliminating the need for additional cloning steps, which makes this cell-free system fast and efficient.
authors.affiliation: Fraunhofer Institute for Biomedical Engineering, Branch Potsdam-Golm, Am Mühlenberg 13, 14476 Potsdam, Germany.
authors.forename: Srujan Kumar
authors.initials: SK
authors.lastname: Dondapati
authors.affiliation: Nanion Technologies GmbH, Gabrielenstr. 9, 80636 Munich, Germany.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Kreir
authors.affiliation: Fraunhofer Institute for Biomedical Engineering, Branch Potsdam-Golm, Am Mühlenberg 13, 14476 Potsdam, Germany.
authors.forename: Robert B
authors.initials: RB
authors.lastname: Quast
authors.affiliation: Fraunhofer Institute for Biomedical Engineering, Branch Potsdam-Golm, Am Mühlenberg 13, 14476 Potsdam, Germany.
authors.forename: Doreen A
authors.initials: DA
authors.lastname: Wüstenhagen
authors.affiliation: Nanion Technologies GmbH, Gabrielenstr. 9, 80636 Munich, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Brüggemann
authors.affiliation: Nanion Technologies GmbH, Gabrielenstr. 9, 80636 Munich, Germany.
authors.forename: Niels
authors.initials: N
authors.lastname: Fertig
authors.affiliation: Fraunhofer Institute for Biomedical Engineering, Branch Potsdam-Golm, Am Mühlenberg 13, 14476 Potsdam, Germany. Electronic address: stefan.kubick@ibmt.fraunhofer.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Kubick
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D008861:Microsomes / Q000378:metabolism; D024642:Potassium Channels, Voltage-Gated / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D055503:Protein Multimerization; D048170:Streptomyces lividans / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D024642:Potassium Channels, Voltage-Gated
keywords: Cell-free; Ion channel; KcsA; Membrane protein expression; Microsome; Single-channel current