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    Characterisation of a cell-free synthesised G-protein coupled receptor

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    9
    Author
    Shilling, PJ; Bumbak, F; Scott, DJ; Bathgate, RAD; Gooley, PR
    Date
    2017-04-24
    Source Title
    Scientific Reports
    Publisher
    NATURE PUBLISHING GROUP
    University of Melbourne Author/s
    Bumbak, Fabian; Scott, Daniel; Bathgate, Ross; Gooley, Paul; Shilling, Patrick
    Affiliation
    Florey Department of Neuroscience and Mental Health
    Biochemistry and Molecular Biology
    School of Chemistry
    Metadata
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    Document Type
    Journal Article
    Citations
    Shilling, P. J., Bumbak, F., Scott, D. J., Bathgate, R. A. D. & Gooley, P. R. (2017). Characterisation of a cell-free synthesised G-protein coupled receptor. SCIENTIFIC REPORTS, 7 (1), https://doi.org/10.1038/s41598-017-01227-z.
    Access Status
    Open Access
    URI
    http://hdl.handle.net/11343/259221
    DOI
    10.1038/s41598-017-01227-z
    NHMRC Grant code
    NHMRC/1081844
    Abstract
    G-protein coupled receptors are the largest family of integral membrane proteins found within the human genome. They function as receptors and modulators to a wide range of ligands and responses which are crucial for human health. GPCR study, specifically the investigation of structure and interaction to cognate ligands, is of high priority. Limitations for structural study can be traced in part, to obtaining suitable quantities of recombinant protein. We sought to address the limitations of traditional recombinant technologies by utilising an Escherichia coli based cell-free protein synthesis (CFPS) approach for production of a thermostable neurotensin receptor 1 (en2NTS1). Initial results were promising, with a high amount (up to 2 mg/mL) of en2NTS1 produced, that had attained correct secondary structure. Meanwhile, concurrent experiments indicated that CFPS produced en2NTS1 showed non-competitive binding to the peptide ligand neurotensin8-13 when compared to E. coli produced en2NTS1. 1H-13C HMQC SOFAST NMR spectra were indicative of disrupted tertiary structure for CFPS produced 13CH3-methionine labelled en2NTS1. The results obtained, indicate CFPS produced en2NTS1 is not forming a discrete tertiary structure and that further development of the CFPS technique needs to be carried out.

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    • School of Chemistry - Research Publications [572]
    • Biochemistry and Molecular Biology - Research Publications [1067]
    • Florey Department of Neuroscience and Mental Health - Research Publications [1312]
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