Functional analysis of cell-free-produced human endothelin B receptor reveals transmembrane segment 1 as an essential area for ET-1 binding and homodimer formation.
Christian Klammt,Ankita Srivastava,Nora Eifler,Friederike Junge,Michael Beyermann,Daniel Schwarz,Hartmut Michel,Volker Doetsch,Frank Bernhard +8 more
TLDR
The quality and functional folding of cell‐free produced human endothelin type B receptor samples are analyzed as an example of the rhodopsin‐type family of G‐protein‐coupled receptors in correlation with different cell‐ free expression modes to provide a valuable tool for fast structural and functional characterizations.Abstract:
The functional and structural characterization of G-protein-coupled receptors (GPCRs) still suffers from tremendous difficulties during sample preparation. Cell-free expression has recently emerged as a promising alternative approach for the synthesis of polytopic integral membrane proteins and, in particular, for the production of G-protein-coupled receptors. We have now analyzed the quality and functional folding of cell-free produced human endothelin type B receptor samples as an example of the rhodopsin-type family of G-protein-coupled receptors in correlation with different cell-free expression modes. Human endothelin B receptor was cell-free produced as a precipitate and subsequently solubilized in detergent, or was directly synthesized in micelles of various supplied mild detergents. Purified cell-free-produced human endothelin B receptor samples were evaluated by single-particle analysis and by ligand-binding assays. The soluble human endothelin B receptor produced is predominantly present as dimeric complexes without detectable aggregation, and the quality of the sample is very similar to that of the related rhodopsin isolated from natural sources. The binding of human endothelin B receptor to its natural peptide ligand endothelin-1 is demonstrated by coelution, pull-down assays, and surface plasmon resonance assays. Systematic functional analysis of truncated human endothelin B receptor derivatives confined two key receptor functions to the membrane-localized part of human endothelin B receptor. A 39 amino acid fragment spanning residues 93-131 and including the proposed transmembrane segment 1 was identified as a central area involved in endothelin-1 binding as well as in human endothelin B receptor homo-oligomer formation. Our approach represents an efficient expression technique for G-protein-coupled receptors such as human endothelin B receptor, and might provide a valuable tool for fast structural and functional characterizations.read more
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Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems
Daniel Schwarz,Friederike Junge,Florian Durst,Nadine Frölich,Birgit Schneider,Sina Reckel,Solmaz Sobhanifar,Volker Dötsch,Frank Bernhard +8 more
TL;DR: This protocol describes the preparation of all essential reaction components such as the E. coli cell extract, T7 RNA polymerase, DNA templates as well as the individual stock solutions for cell-free expression.
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Amphipols From A to Z
J-L Popot,Thorsten Althoff,D Bagnard,J-L Banères,Paola Bazzacco,Emmanuelle Billon-Denis,Laurent J. Catoire,Philippe Champeil,Delphine Charvolin,Melanie J. Cocco,G Crémel,Tassadite Dahmane,L M de la Maza,Christine Ebel,F Gabel,Fabrice Giusti,Yann Gohon,Erik Goormaghtigh,Emmanuel-Pierre Guittet,Jörg H. Kleinschmidt,Werner Kühlbrandt,C. Le Bon,Karen L. Martinez,Melanie Picard,B Pucci,Jonathan N. Sachs,Christophe Tribet,C. van Heijenoort,Frank Wien,Francesca Zito,Manuela Zoonens +30 more
TL;DR: Applications of APols include MP folding and cell-free synthesis, structural studies by NMR, electron microscopy and X-ray diffraction, APol-mediated immobilization of MPs onto solid supports, proteomics, delivery of MPs to preexisting membranes, and vaccine formulation.
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Large-scale production of functional membrane proteins
TL;DR: The review will give an overview of currently established processes for the production of functionally folded membrane proteins, a highly dynamic field where new or modified approaches are frequently emerging.
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Membrane protein expression: no cells required
TL;DR: The most recent advances in cell-free protein expression are reviewed, underscoring the potentials and weaknesses of the newly developed approaches and place specific emphasis on the use of nanolipoprotein particles (NLPs or nanodiscs).
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Production of membrane proteins using cell-free expression systems.
TL;DR: Cell‐free (CF) expression systems have been developed in recent times as promising tools by offering completely new approaches to synthesize MPs directly into artificial hydrophobic environments with high success rates and rapidly accumulating data on quality and expression efficiencies.
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