Browsing by Author "Tait, Timo"

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    The production and purification of functional steroid hormone receptor ligand binding domains towards the development of a biological endocrine disruptor detection system
    (Stellenbosch : Stellenbosch University, 2015-04) Tait, Timo; Swart, Pieter; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: During the last two and a half decades a large body of research has accumulated indicating the presence of various natural and synthetic chemical compounds within the environment capable of inducing hormone-like responses in humans and animals. Such compounds, termed endocrine disruptors, have been implicated in a variety of developmental, reproductive and physiological abnormalities which have been shown to converge on the endocrine system. Given that endocrine disrupters are comprised of a diverse group of molecules with dissimilar chemical structures, general screening techniques are not feasible for effective environmental monitoring. A primary method of action by which these exogenous molecules affect the homeostatic regulation of the endocrine system is believed to be via the modulation of gene transcription. It is now well established that many endocrine disrupting compounds act upon a principal group of transcription factors, the nuclear receptors, by chance interaction with the ligand binding domains of these proteins. With a view to ultimately design a portable kit for the detection of endocrine disrupting compounds in water based on the bio-specific immobilisation of nuclear receptor ligand binding domains to a stationary membrane matrix, this study specifically describes: 1. The effects on recombinant protein expression by the addition of small molecules to the cultivation media of bacteria. 2. The optimisation of conditions for the lysis of bacterial cells to increase the solubility of heterologously expressed proteins. 3. The purification of recombinant proteins from bacterial cell lysates by means of a two-step chromatographic methodology. 4. The cloning of the genes for the human androgen and estrogen receptors’ ligand binding domains into baculovirus transfer plasmids. 5. Transfer of genetic material from the created baculovirus transfer plasmids to a linearised baculovirus genome for the generation of recombinant viruses. 6. The cultivation, and baculoviral infection, of Spodoptera frugiperda and Trichoplusia ni cell lines. 7. Expression and purification of N-terminal hexahistidine-tagged human nuclear receptor LBDs from insect cell lysates by means of immobilised metal affinity chromatography.
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    Strategies for the detection of endocrine disrupting compounds utilising recombinant oestrogen receptor ligand-binding domains and high specificity monoclonal antibodies
    (Stellenbosch : Stellenbosch University, 2018-12) Tait, Timo; Swart, Pieter; Swart, Amanda C.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: During the last two and a half decades, a large body of research has been amassed which indicate that various compounds capable of inducing hormone-like responses in humans and animals are present within the environment. These natural and synthetic molecules, termed endocrine disruptors, have been implicated in a variety of developmental, reproductive and physiological abnormalities which have been shown to converge on the endocrine system. Given that endocrine disrupters are comprised of a diverse group of molecules with dissimilar chemical structures, general screening techniques are not feasible for effective environmental monitoring. A primary method of action by which these exogenous molecules affect the homeostatic regulation of the endocrine system is believed to be via the modulation of gene transcription. It is now well established that many endocrine disrupting compounds act upon a principal group of transcription factors, the nuclear receptors, by chance interaction with the ligand-binding domains of these proteins. The current consensus is that endocrine disruptors pose a significant, long-term environmental risk to the well-being of both humans and wildlife. Resultantly, there is a growing need for the development of technologies that can be employed in the rapid assessment of environmental samples for the presence of detrimental xenobiotics. In the view of this need, this dissertation describes membrane-based biological devices which may ultimately form the basis of novel assays which may be used for the rapid detection of endocrine disrupting compounds in remote geographical locations. Specifically, this study describes: 1. A review of the development of in vitro membrane-based immunoassays used in rapid diagnostics, point-of-care analysis and over-the-counter devices for the detection of diseases, environmental contaminants, drugs of abuse and adulterants in food and water supplies. 2. The heterologous expression and purification of highly functional recombinant forms of the alpha isoform of the human oestrogen receptor ligand-binding domain. 3. The characterisation of the ligand-binding properties of these receptor molecules regarding the natural ligand, 17β-oestradiol, other hormones and several known endocrine disruptors by saturation and competitive radioligand-binding methods. 4. Immobilisation of the receptor proteins onto a synthetic support via chelation chemistry and the determination of endocrine active compound sequestration to the biofunctionalised membrane. 5. The establishment, selection and propagation of immortal murine hybridoma cell strains derived from antigen-stimulated B-cells fused to Sp2/0-Ag14 murine myeloma cells. 6. The purification of monoclonal antibodies raised against a defined epitope on the human oestrogen receptor alpha ligand-binding domain surface. 7. Covalent labelling of purified monoclonal antibodies with colloidal gold nanoparticles in preparation for the development of a novel immunochromatographic device.