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    Cell-free synthesis of isotopically labelled peptide ligands for the functional characterization of G protein-coupled receptors

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    Author
    Joedicke, L; Trenker, R; Langer, JD; Michel, H; Preu, J
    Date
    2016-01-01
    Source Title
    FEBS Open Bio
    Publisher
    WILEY-BLACKWELL
    University of Melbourne Author/s
    Trenker, Raphael
    Affiliation
    Medical Biology (W.E.H.I.)
    Metadata
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    Document Type
    Journal Article
    Citations
    Joedicke, L., Trenker, R., Langer, J. D., Michel, H. & Preu, J. (2016). Cell-free synthesis of isotopically labelled peptide ligands for the functional characterization of G protein-coupled receptors. FEBS OPEN BIO, 6 (1), pp.90-102. https://doi.org/10.1002/2211-5463.12008.
    Access Status
    Open Access
    URI
    http://hdl.handle.net/11343/259086
    DOI
    10.1002/2211-5463.12008
    Abstract
    Cell-free systems exploit the transcription and translation machinery of cells from different origins to produce proteins in a defined chemical environment. Due to its open nature, cell-free protein production is a versatile tool to introduce specific labels such as heavy isotopes, non-natural amino acids and tags into the protein while avoiding cell toxicity. In particular, radiolabelled peptides and proteins are valuable tools for the functional characterization of protein-protein interactions and for studying binding kinetics. In this study we evaluated cell-free protein production for the generation of radiolabelled ligands for G protein-coupled receptors (GPCRs). These receptors are seven-transmembrane-domain receptors activated by a plethora of extracellular stimuli including peptide ligands. Many GPCR peptide ligands contain disulphide bonds and are thus inherently difficult to produce in bacterial expression hosts or in Escherichia coli-based cell-free systems. Here, we established an adapted E. coli-based cell-free translation system for the production of disulphide bond-containing GPCR peptide ligands and specifically introduce tritium labels for detection. The bacterial oxidoreductase DsbA is used as a chaperone to favour the formation of disulphide bonds and to enhance the yield of correctly folded proteins and peptides. We demonstrate the correct folding and formation of disulphide bonds and show high-affinity ligand binding of the produced radio peptide ligands to the respective receptors. Thus, our system allows the fast, cost-effective and reliable synthesis of custom GPCR peptide ligands for functional and structural studies.

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