• myGriffith
    • Staff portal
    • Contact Us⌄
      • Future student enquiries 1800 677 728
      • Current student enquiries 1800 154 055
      • International enquiries +61 7 3735 6425
      • General enquiries 07 3735 7111
      • Online enquiries
      • Staff phonebook
    View Item 
    •   Home
    • Griffith Theses
    • Theses - Higher Degree by Research
    • View Item
    • Home
    • Griffith Theses
    • Theses - Higher Degree by Research
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Browse

  • All of Griffith Research Online
    • Communities & Collections
    • Authors
    • By Issue Date
    • Titles
  • This Collection
    • Authors
    • By Issue Date
    • Titles
  • Statistics

  • Most Popular Items
  • Statistics by Country
  • Most Popular Authors
  • Support

  • Contact us
  • FAQs
  • Admin login

  • Login
  • Thioredoxin and Oxidative Stress

    Thumbnail
    View/Open
    02Whole.pdf (3.281Mb)
    Author(s)
    Gregory, Sarah-Jane S.
    Primary Supervisor
    Perkins, Tony
    Year published
    2004
    Metadata
    Show full item record
    Abstract
    The experiments described in this thesis involve the expression and characterisation of recombinant truncated thioredoxin (tTrx) and the potential involvement that thioredoxin (Trx) has in the cellular responses to oxidative stress. Truncated Trx (80 amino acids) was expressed from a plasmid containing the ORF for tTrx that had been introduced into E.coli BL-21(DE3) cells. The protein was initially extracted using a combination of high concentrations of urea, high pH levels, and multiple sonification steps to remove the tTrx from inclusion bodies formed during expression. This procedure produced a stable solution of tTrx. ...
    View more >
    The experiments described in this thesis involve the expression and characterisation of recombinant truncated thioredoxin (tTrx) and the potential involvement that thioredoxin (Trx) has in the cellular responses to oxidative stress. Truncated Trx (80 amino acids) was expressed from a plasmid containing the ORF for tTrx that had been introduced into E.coli BL-21(DE3) cells. The protein was initially extracted using a combination of high concentrations of urea, high pH levels, and multiple sonification steps to remove the tTrx from inclusion bodies formed during expression. This procedure produced a stable solution of tTrx. Purification of tTrx from this protein solution required anion exchange chromatography followed by gel permeation in a HPLC system to obtain fully purified, recombinant tTrx which allowed further characterisation studies to be undertaken. An initial investigation into tTrx was performed to determine some basic physical, biochemical and functional aspects of this hitherto relatively undefined protein. Analysis by sedimentation equilibrium indicated that freshly prepared tTrx forms a single species with a molecular weight of 18.8kDa. This value indicates that recombinant tTrx naturally forms a dimer in solution that was shown to be non-covalent in nature and stable in solution. The capacity of tTrx to reduce protein disulphide bonds was determined using the insulin reduction assay. Results show that tTrx lacks this particular redox ability. The rate of oxidisation at 4 degrees C was analysed using free thiol determination, sedimentation equilibrium and SDS-PAGE patterning. Results indicated a steady rise in the degree of oxidation of tTrx over an eight day period. After six days the oxidated protein consistently displayed the presence of intramolecular disulphide bonds. Covalently-linked disulphide dimers and higher molecular weight oligomers were detectable after eight days oxidation. An investigation of the reducing capacity of the basic Trx system determined that fully oxidised tTrx was unable to act alone as a substrate for thioredoxin reductase (TR). However, when reduced Trx was added to the system, it appeared capable of acting as an electron donor to the oxidised tTrx in order to reduce disulphide groups. Recombinant tTrx was successfully radiolabelled with Trans 35S-methionine/cysteine for use in cell association studies. No evidence was found to indicate the presence of a receptor for tTrx on either MCF-7 or U-937 cells. Findings suggest that a low level of non-specific binding of tTrx to these cell lines rather than a classical ligand-binding mechanism occurs thus suggesting the absence of a cell surface receptor for tTrx. The role that Trx may play in the cellular responses to oxidative stress was also investigated. The chemical oxidants hydrogen peroxide (H2O2) and diamide were used to establish an in vitro model of oxidative stress for the choriocarcinoma cytotrophoblast cell line JEG-3. Cellular function was assessed in terms of membrane integrity, metabolic activity and the ability to synthesis new DNA following exposure to these oxidants. Results indicated that both agents were capable of causing cells to undergo oxidative stress without inducing immediate apoptosis or necrosis. Initially, JEG-3 cells exposed to 38μM or 75μM H2O2 or 100μM diamide were shown to display altered cell metabolism and DNA synthesis without loss to cell viability or membrane integrity. Cells were also shown to be capable of some short-term recovery but later lapsed into a more stressed state. Expression levels of Trx were studied to determine whether this type of chemical stress caused a change in intercellular protein levels. Both cELISA and western blotting results indicated that only cells exposed to 100μM diamide displayed any significant increase in Trx protein levels after 6 or 8hrs exposure to the oxidant. Further studies over a longer time-frame were also performed. These found that when JEG-3 cells were exposed to 18μM H2O2 or 200μM diamide over 12-48hrs, a positive correlation between increasing endogenous Trx protein levels and a decline in cell proliferation was observed. Cytotrophoblast cells, which are responsible for implantation and placentation, are susceptible to oxidative stress in vivo and their anti-oxidant capacity is fundamental to the establishment of pregnancy. The findings obtained during these studies suggest that Trx plays a role in this process.
    View less >
    Thesis Type
    Thesis (Masters)
    Degree Program
    Master of Philosophy (MPhil)
    School
    School of Health Sciences
    DOI
    https://doi.org/10.25904/1912/3680
    Copyright Statement
    The author owns the copyright in this thesis, unless stated otherwise.
    Item Access Status
    Public
    Subject
    recombinant truncated thioredoxin
    tTrx
    oxidative stress
    oxidisation
    oxidization
    protein
    proteins
    chemistry
    biochemistry
    Publication URI
    http://hdl.handle.net/10072/367183
    Collection
    • Theses - Higher Degree by Research

    Footer

    Disclaimer

    • Privacy policy
    • Copyright matters
    • CRICOS Provider - 00233E
    • TEQSA: PRV12076

    Tagline

    • Gold Coast
    • Logan
    • Brisbane - Queensland, Australia
    First Peoples of Australia
    • Aboriginal
    • Torres Strait Islander