title: Cell-free production of VDAC directly into liposomes for integration with biomimetic membrane systems.
abstract: The mitochondrial voltage-dependent anion channel (VDAC) is a pivotal protein since it provides the major transport pathway between the cytosol and the mitochondrial intermembrane space and it is implicated in cell apoptosis by functioning as a gatekeeper for the trafficking of mitochondrial death molecules. VDAC is a beta-barrel channel with a large conductance, and we use it as a model transport protein for the design of biomimetic systems. To overcome the limitations of classical overexpression methods for producing and purifying membrane proteins (MPs) we describe here the use of an optimized cell-free system. In a one-step reaction VDAC is obtained directly integrated into liposomes and purified by ultracentrifugation. We then combine proteoliposomes with different bilayers models in order to validate VDAC insertion and functionality. This VDAC biomimetic model is the first example validating the use of a cell-free expression system for production of MPs into liposomes and tethered bilayers as a toolbox to build a wide range of biomimetic devices.
authors.affiliation: a University of Grenoble 1-Joseph Fourier/CNRS/TIMC-IMAG UMR 5525 (Equipe SyNaBi) , Grenoble , France.
authors.forename: Lavinia
authors.initials: L
authors.lastname: Liguori
authors.affiliation: a University of Grenoble 1-Joseph Fourier/CNRS/TIMC-IMAG UMR 5525 (Equipe SyNaBi) , Grenoble , France.
authors.forename: Barry
authors.initials: B
authors.lastname: Stidder
authors.affiliation: a University of Grenoble 1-Joseph Fourier/CNRS/TIMC-IMAG UMR 5525 (Equipe SyNaBi) , Grenoble , France.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Alcaraz
authors.affiliation: a University of Grenoble 1-Joseph Fourier/CNRS/TIMC-IMAG UMR 5525 (Equipe SyNaBi) , Grenoble , France.
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Lenormand
authors.affiliation: a University of Grenoble 1-Joseph Fourier/CNRS/TIMC-IMAG UMR 5525 (Equipe SyNaBi) , Grenoble , France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Cinquin
authors.affiliation: a University of Grenoble 1-Joseph Fourier/CNRS/TIMC-IMAG UMR 5525 (Equipe SyNaBi) , Grenoble , France.
authors.forename: Donald K
authors.initials: DK
authors.lastname: Martin
mesh_terms: D032701:Biomimetics; D015153:Blotting, Western; D002474:Cell-Free System; D002942:Circular Dichroism; D003001:Cloning, Molecular; D008081:Liposomes; D008567:Membranes, Artificial; D016253:Microscopy, Immunoelectron; D050994:Voltage-Dependent Anion Channels / Q000235:genetics / Q000378:metabolism*
chemical_list: D008081:Liposomes; D008567:Membranes, Artificial; D050994:Voltage-Dependent Anion Channels
keywords: Biomimetic devices; VDAC liposomes; cell-free expression system; electrical measurements; membrane proteins