Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter |
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Authors: | Kamila Pszon-Bartosz,Jesper S. Hansen,Karin B. Stibius,Jesper S. Groth,Jenny Emné us,Oliver Geschke,Claus Hé lix-Nielsen |
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Affiliation: | aAquaporin A/S, Ole Maaløes Vej 3, DK-2200 Copenhagen N, Denmark;bTechnical University of Denmark, Department of Micro- and Nanotechnology, DK-2800 Kongens Lyngby, Denmark;cTechnical University of Denmark, Department of Physics, DTU Physics, DK-2800 Kongens Lyngby, Denmark |
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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 105 FomA proteins could be incorporated in a bilayer array with a total membrane area of 2 mm2 within 20 min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications. |
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Keywords: | Abbreviations: BFS, bilayer forming solution BLM, black lipid membrane DOPC, 1,2-dioloeyl-sn-glycero-3-phosphocholine DPhPC, 1,2-diphytanoyl-sn-glycero-3-phosphocholine ETFE, ethylene tetrafluoroethylene FomA, major outer membrane protein of Fusobacterium nucleatum LDAO, N-lauryl-N,N-dimethylammonium-N-oxide LPR, lipid to protein molar ratio NBD-PC, 1-oleoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phosphocholine NTA, nanoparticle tracking analysis OG, octyl-β- font-variant: small-caps" >d-glucopyranoside PBS, phosphate buffered saline PEG, polyethylene glycol SNAP, soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) SNARE, SNAP receptor protein Texas Red® , sulforhodamine 101 acid chloride VDAC, mitochondrial voltage dependent anion channel |
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