Production and Enzymatic Degradation of Sup35NM, a Prion-Like Protein from Yeast
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Date
2005-08-03
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Abstract
Prion diseases, or transmissible spongiform encephalopathies (TSEs), including human Creutzfeld-Jakob disease (CJD) and bovine spongiform encephalopathy (BSE), are fatal neurodegenerative diseases of humans and animals that are caused by aggregates of the inheritable, protease-resistant, self-propagating isoform of prion proteins, PrPSc.
A bacterial keratinase produced by Bacillus licheniformis strain PWD-1 was found to be able to degrade this prion under certain conditions; however, since this disease-causing prion is infectious and expensive to work with, a model or surrogate protein was needed for laboratory studies. This study developed the use of a yeast prion protein system, the prion-determining NM region of the yeast prion protein Sup35p from Saccharomyces cerevisiae, that is structurally similar, but non-pathogenic, to study the enzymatic degradative process. Sup35NM was expressed and purified from Escherichia coli to form amyloid in vitro, a phenomenon similar to the amyloid formation in vivo in CJD and BSE. Aggregation and de-aggregation of Sup35NM were examined by gel electrophoresis, Congo red binding, fluorescence, Western blot and electron microscopy. Proteinase K (PK) and keratinase (KE) were used to characterize the yeast prion, Sup35NM. Protease concentrations, preheating temperatures, reaction temperatures, and reaction times were studied to characterize its enzymatic digestion patterns. Consequently, optimal conditions and other active enzymes can be further tested with BSE tissues and prions. In the long run, an effective enzymatic process can be developed to inactivate prions in food and feed to prevent the spread of TSEs.
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prion, enzymatic degradation, keratinase, Sup35NM, Sup35, yeast prion
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Degree
MS
Discipline
Microbiology
