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  • Expression, solubilisation, and purification of a functional CMP-sialic acid transporter in Pichia pastoris

    Author(s)
    Maggioni, Andrea
    Hadley, Barbara
    von Itzstein, Mark
    Tiralongo, Joe
    Griffith University Author(s)
    von Itzstein, Mark
    Maggioni, Andrea
    Tiralongo, Joe
    Hadley, Barbara J.
    Year published
    2014
    Metadata
    Show full item record
    Abstract
    Membrane proteins, including solute transporters play crucial roles in cellular function and have been implicated in a variety of important diseases, and as such are considered important targets for drug development. Currently the drug discovery process is heavily reliant on the structural and functional information discerned from high-resolution crystal structures. However, membrane protein structure determination is notoriously difficult, due in part to challenges faced in their expression, solubilisation and purification. The CMP-sialic acid transporter (CST) is considered to be an attractive target for drug discovery. ...
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    Membrane proteins, including solute transporters play crucial roles in cellular function and have been implicated in a variety of important diseases, and as such are considered important targets for drug development. Currently the drug discovery process is heavily reliant on the structural and functional information discerned from high-resolution crystal structures. However, membrane protein structure determination is notoriously difficult, due in part to challenges faced in their expression, solubilisation and purification. The CMP-sialic acid transporter (CST) is considered to be an attractive target for drug discovery. CST inhibition reduces cancer cell sialylation and decreases the metastatic potential of cancer cells and to date, no crystal structure of the CST, or any other nucleotide sugar transporter exists. Here we describe the optimised conditions for expression in Pichia pastoris, solubilisation using n-nonyl ߭d-maltopyranoside (NM) and single step purification of a functional CST. Importantly we show that despite being able to solubilise and purify the CST using a number of different detergents, only NM was able to maintain CST functionality.
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    Journal Title
    Protein Expression Purification
    Volume
    101
    DOI
    https://doi.org/10.1016/j.pep.2014.07.003
    Subject
    Biochemistry and cell biology
    Receptors and membrane biology
    Other biological sciences
    Publication URI
    http://hdl.handle.net/10072/66894
    Collection
    • Journal articles

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