Characterisation of Transgenic Tobacco Plants Expressing Synthetic Mouse Prion Protein

PhD Thesis


Nworji, Ogechukwu Frances 2016. Characterisation of Transgenic Tobacco Plants Expressing Synthetic Mouse Prion Protein. PhD Thesis University of East London Health, Sport and Bioscience
AuthorsNworji, Ogechukwu Frances
TypePhD Thesis
Abstract

The cellular prion protein (PrPC) is a glycoprotein with unknown function constitutively expressed in mammalian neurons. PrPC converts to a pathogenic misfolded isomer (PrPSc) through a poorly understood process, resulting in a group of fatal neurodegenerative diseases collectively known as transmissible spongiform encephalopathy or prion disease. Elucidating the molecular mechanisms behind prion conversion requires production of PrPC in recombinant systems. This study was designed to generate transgenic tobacco plants expressing recombinant mouse prion protein (mPrP). Using a synthetic gene encoding the mouse prion protein, plant expression vectors were constructed for constitutive mPrP expression in the apoplast (pGreen35SmPrP-Apo), cytosol (pGreen35SmPrP-Cyto) and endoplasmic reticulum (pGreen35SmPrP-ER). Putative transgenic plants transformed with either pGreen35SmPrP-Cyto or pGreen35SmPrP-ER were analysed by PCR, ELISA and immunoblot for transgene integration and expression. However, no viable plants were obtained from the pGreen35SmPrP-Apo transformants. ELISA analysis showed that recombinant mPrP accumulated up to 0.0024% of total soluble leaf protein in transgenic tobacco leaves transformed with the pGreen35SmPrP-Cyto construct and 0.0016% of total soluble leaf protein in plants designed to sequester recombinant mPrP to the ER. Furthermore, immunoblot analysis showed that ER-targeted recombinant mPrP was mainly unglycosylated, although a glycosylated mPrP isoform was observed indicating that transgenic tobacco plants process ER-targeted recombinant mPrP in a manner analogous to mammalian systems. The nutrient composition of several transgenic plants were analysed to determine the phenotypic effect of expressing recombinant mPrP in tobacco plants. The analysis revealed that transgenic lines expressing cytosolic-mPrP had elevated average levels of Mn2+ and Fe2+. In addition, kanamycin-treated transgenic plants expressing cytosolic-mPrP developed a non-rooting phenotype. Conversely, the average Cu2+ level was increased in analysed transgenic plants designed to sequester recombinant mPrP in the ER. Furthermore, the plants developed no visible phenotype upon kanamycin treatment. This result support studies that suggest that the PrPC has functional role in metal homeostasis and loss of its thermodynamic structure leads to metal dyshomeostasis which in turn has been linked to prion disease associated neurotoxicity. Finally, the recombinant mPrP was purified via affinity chromatography facilitated by the presence of a C-terminal polyhistidine tag on the synthetic gene constructs.

Year2016
Digital Object Identifier (DOI)doi:10.15123/PUB.5838
Publication dates
PrintSep 2016
Publication process dates
Deposited24 Apr 2017
Publisher's version
License
CC BY-NC-ND
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