title: Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter.
abstract: Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR)=50 more than 10(5) FomA proteins could be incorporated in a bilayer array with a total membrane area of 2mm(2) within 20 min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications.
authors.affiliation: Aquaporin A/S, Ole Maaløes Vej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Kamila
authors.initials: K
authors.lastname: Pszon-Bartosz
authors.forename: Jesper S
authors.initials: JS
authors.lastname: Hansen
authors.forename: Karin B
authors.initials: KB
authors.lastname: Stibius
authors.forename: Jesper S
authors.initials: JS
authors.lastname: Groth
authors.forename: Jenny
authors.initials: J
authors.lastname: Emnéus
authors.forename: Oliver
authors.initials: O
authors.lastname: Geschke
authors.forename: Claus
authors.initials: C
authors.lastname: Hélix-Nielsen
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008561:Membrane Fusion; D040081:Protein Array Analysis / Q000379:methods*; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism; D050994:Voltage-Dependent Anion Channels / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D008051:Lipid Bilayers; D011510:Proteolipids; D050994:Voltage-Dependent Anion Channels; C083760:fomA protein, Fusobacterium; C020485:proteoliposomes