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  • Protein engineering of streptavidin for in vivo assembly of streptavidin beads

    Author(s)
    Peters, Verena
    Rehm, Bernd HA
    Griffith University Author(s)
    Rehm, Bernd
    Year published
    2008
    Metadata
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    Abstract
    Escherichia coli was engineered to intracellularly manufacture streptavidin beads. Variants of streptavidin (monomeric, core and mature full length streptavidin) were C-terminally fused to PhaC, the polyester granule forming enzyme of Cupriavidus necator. All streptavidin fusion proteins mediated formation of the respective granules in E. coli and were overproduced at the granule surface. The monomeric streptavidin showed biotin binding (0.7 ng biotin/μg bead protein) only when fused as single-chain dimer. Core streptavidin and the corresponding single-chain dimer mediated a biotin binding of about 3.9 and 1.5 ng biotin/μg ...
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    Escherichia coli was engineered to intracellularly manufacture streptavidin beads. Variants of streptavidin (monomeric, core and mature full length streptavidin) were C-terminally fused to PhaC, the polyester granule forming enzyme of Cupriavidus necator. All streptavidin fusion proteins mediated formation of the respective granules in E. coli and were overproduced at the granule surface. The monomeric streptavidin showed biotin binding (0.7 ng biotin/μg bead protein) only when fused as single-chain dimer. Core streptavidin and the corresponding single-chain dimer mediated a biotin binding of about 3.9 and 1.5 ng biotin/μg bead protein, respectively. However, biotin binding of about 61 ng biotin/μg bead protein with an equilibrium dissociation constant (KD) of about 4 × 10−8 M was obtained when mature full length streptavidin was used. Beads displaying mature full length streptavidin were characterized in detail using ELISA, competitive ELISA and FACS. Immobilisation of biotinylated enzymes or antibodies to the beads as well as the purification of biotinylated DNA was used to demonstrate the applicability of these novel streptavidin beads. This study proposes a novel method for the cheap and efficient one-step production of versatile streptavidin beads by using engineered E. coli as cell factory.
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    Journal Title
    Journal of Biotechnology
    Volume
    134
    Issue
    3-4
    DOI
    https://doi.org/10.1016/j.jbiotec.2008.02.006
    Subject
    Biological sciences
    Biochemistry and cell biology not elsewhere classified
    Engineering
    Publication URI
    http://hdl.handle.net/10072/371093
    Collection
    • Journal articles

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