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  • Direct detection of ligand binding to Sepharose-immobilised protein using saturation transfer double difference (STDD) NMR spectroscopy

    Author(s)
    Haselhorst, Thomas
    Muenster-Kuehnel, Anja K
    Oschlies, Melanie
    Tiralongo, Joe
    Gerardy-Schahn, Rita
    von Itzstein, Mark
    Griffith University Author(s)
    von Itzstein, Mark
    Haselhorst, Thomas E.
    Tiralongo, Joe
    Oschlies, Melanie
    Year published
    2007
    Metadata
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    Abstract
    We report an easy and direct application of 'Saturation Transfer Double Difference' (STDD) NMR spectroscopy to identify ligands that bind to a Sepharose-immobilised target protein. The model protein, cytidine 5'-monophosphate sialic acid (CMP-Sia) synthetase, was expressed as a Strep-Tag II fusion protein and immobilised on Strep-Tactinepharose. STD NMR experiments of the protein-enriched Sepharose matrix in the presence of a binding ligand (cytidine 5'-triphosphate, CTP) and a non-binding ligand (a/߭glucose) clearly show that CTP binds to the immobilised enzyme, whereas glucose has no affinity. This approach has three major ...
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    We report an easy and direct application of 'Saturation Transfer Double Difference' (STDD) NMR spectroscopy to identify ligands that bind to a Sepharose-immobilised target protein. The model protein, cytidine 5'-monophosphate sialic acid (CMP-Sia) synthetase, was expressed as a Strep-Tag II fusion protein and immobilised on Strep-Tactinepharose. STD NMR experiments of the protein-enriched Sepharose matrix in the presence of a binding ligand (cytidine 5'-triphosphate, CTP) and a non-binding ligand (a/߭glucose) clearly show that CTP binds to the immobilised enzyme, whereas glucose has no affinity. This approach has three major advantages: (a) only low quantities of protein are required, (b) no specialised NMR technology or the application of additional data analysis by non-routine methods is required, and (c) easy multiple use of the immobilised protein is available.
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    Journal Title
    Biochemical and Biophysical Research Communications
    Volume
    359
    Publisher URI
    http://www.elsevier.com/wps/find/journaldescription.cws_home/622790/description#description
    DOI
    https://doi.org/10.1016/j.bbrc.2007.05.204
    Copyright Statement
    © 2007 Elsevier. Please refer to the journal's website for access to the definitive, published version.
    Subject
    Medicinal and biomolecular chemistry
    Biochemistry and cell biology
    Medical biochemistry and metabolomics
    History, heritage and archaeology
    Publication URI
    http://hdl.handle.net/10072/18291
    Collection
    • Journal articles

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