Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes |
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Authors: | Susanne F Fenz Rita Sachse Thomas Schmidt Stefan Kubick |
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Institution: | 1. Leiden Institute of Physics, Leiden University, PO Box 9504, 2300 RA Leiden, The Netherlands;2. Fraunhofer IBMT, Branch Potsdam-Golm, Group of Cell-free Protein Synthesis, Am Mühlenberg 13, 14476 Potsdam, Germany |
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Abstract: | Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces. |
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Keywords: | GUVs Giant unilamellar vesicles GPCR G protein-coupled receptor eYFP Enhanced yellow fluorescent protein ETB Endothelin B receptor CXCR4 C-X-C chemokine receptor type 4 Hb-EGF Pro-Heparin-binding epithelial growth factor like factor ER Endoplasmic reticulum E-PCR Expression polymerase chain reaction TCA Trichloroacetic acid TMD Transmembrane domain DOPC 1 2-dioleoyl-sn-glycero-3-phosphocholine ITO Indium tin oxide DOPE biotin 1 2-dioleoyl-sn-glycero-3-phospho-ethanolamine-N-(cap biotinyl) msd Mean squared-displacement D Diffusion coefficient E coli Escherichia coli exp Experimental value theo Theoretical value CFP Cyan fluorescent protein PDMS Polydimethylsiloxane δ One-dimensional localization precision D Diffusion coefficient n Number of integrated target membrane proteins N Number of single molecules detected per movie μ&prime Medium's viscosity μ Membrane's viscosity kB Boltzmann's constant T Temperature b mobility h Membrane thickness ε Reduced radius |
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