The production and purification of functional steroid hormone receptor ligand binding domains towards the development of a biological endocrine disruptor detection system

Tait, Timo (2015-04)

Thesis (MSc)--Stellenbosch University, 2015.

Thesis

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.

AFRIKAANSE OPSOMMING: Die teenwoordigheid van natuurlike en sintetiese chemiese middels wat oor die vermoë beskik om die aksies van hormone in die mens en dier na te boots het toenemend aftrek gekry in navorsing gedurende die laaste twee en ’n halwe dekades. 'n Verskeidenheid van ontwikkelings-, reproduktiewe- en fisiologiese abnormaliteite ontstaan as gevolg van die aksies van hierdie molekule, genaamd endokriene-ontwrigters, op die natuurlike funksionering van die endokriene-sisteem. Gegewe dat die groep chemiese middels waaruit endokriene-ontwrigters bestaan van diverse oorsprong afkomstig is lei dit daartoe dat algemene analitiese tegnieke nie in alle gevalle geskik is vir effektiewe omgewingsmonitering is nie. Die modulasie van geentranskripsie is een van die metodes wat voorgestel word as ’n metode waarop hierdie eksogene molekule die homeostatiese regulering deur die endokriene-sisteem omverwerp. ’n Algemene metode waarop vele endokrien-ontwrigtende stowwe geentranskripsie beïnvloed, is deur interaksie met die hormoon-bindende gedeeltes van ’n belangrike groep transkripsiefaktore, die nukluêre reseptore. Hierdie studie, met die uiteindelike ontwikkeling van ’n draagbare toetsstelsel vir die opsporing van endokrien-ontwrigtende-stowwe in water, gebasseer op die bio-spesifieke immobilisering van nukluêre reseptor ligand bindingsdomeins op ’n stasionêre membraanmatriks, het ten doel om die volgende te beskryf: 1. Die effek wat die byvoeging van klein molekule tot die groeimedium van bakteriëe het op die uitdrukking van rekombinante proteïene. 2. Die optimisering van bakteriese sel-lisering in terme van verhoging in die oplosbaarheid van heteroloë proteïene. 3. Die suiwering van rekombinante proteïen vanuit bakteriese sellisate deur middel van ’n twee-stap chromatografiese sisteem. 4. Die klonering van die gene vir die menslike androgeen en estrogeen reseptore se ligand bindingsdomeine in bakulovirus oordragplasmiede. 5. Die oordrag van genetiese materiaal vanaf hierdie bakulovirus oordragplasmiede na ’n gelineariseerde bakulovirus genoom deur middel van homoloë rekombinasie vir die produksie van rekombinante virusse. 6. Die groei en infeksie van Spodoptera frugiperda en Trichoplusia ni sellyne wat lei tot die uitdrukking van menssoortgelyke nukluêre reseptor ligandbindingsdomains. 7. Suiwering van N-terminaal heksahistidien-etiket-gekoppelde menslike nukluêre reseptor ligandbindingsdomeins vanuit inseksellisate deur middel van geïmmobiliseerde metaal affiniteitschromatografie.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/96811
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