Expression optimization and NMR spectroscopy of Giardia intestinalis cytochrome b5 isotype III

Abstract

The parasitic protist Giardia intestinalis does not synthesize heme and lacks many common eukaryotic heme proteins, yet it expresses four cytochrome b5 (gCYTB5) isotypes of unknown function. These have low reduction potentials and distinct subcellular locations that are consistent with structural features and biological functions that differ from their mammalian counterparts. Isotype III (gCYTB5-III) is particularly fascinating for its unusual location in the nuclei of Giardia. This thesis reports the optimization of recombinant gCYTB5-III overexpression for structural studies by NMR spectroscopy. Vital optimization factors for isotope labelling were first identified, finding that auto-induction promotes the optimization of many other conditions, such as colony selection, starter cultures, media components, temperature, pH and aeration. Optimized conditions were then applied to the expression and NMR spectroscopy of isotope-labelled gCYTB5-III and bovine cytochrome b5 as a control. These results can be extended to other heme proteins and will expand our biochemical knowledge of Giardia.

Author Keywords: Auto-induction, Cytochrome b5, Giardia intestinalis, Isotope Labelling, Nuclear Magnetic Resonance Spectroscopy, Recombinant Protein

    Item Description
    Type
    Contributors
    Creator (cre): Crowley, Erika Lynn
    Thesis advisor (ths): Rafferty, Steven P
    Degree committee member (dgc): Vreugdenhil, Andrew
    Degree committee member (dgc): Brunetti, Craig
    Degree granting institution (dgg): Trent University
    Date Issued
    2019
    Date (Unspecified)
    2019
    Place Published
    Peterborough, ON
    Language
    Extent
    106 pages
    Rights
    Copyright is held by the author, with all rights reserved, unless otherwise noted.
    Subject (Topical)
    Local Identifier
    TC-OPET-10746
    Publisher
    Trent University
    Degree
    Master of Science (M.Sc.): Environmental and Life Sciences