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Monitoring Human Copper/Zinc Superoxide Dismutase Misfolding/Aggregation in Mammalian Cells

Gregoire, Simpson
Format
Thesis/Dissertation; Online
Author
Gregoire, Simpson
Advisor
Kwon, Inchan
Abstract
Protein misfolding and aggregation play important roles in many processes in living organisms. These include pathological protein aggregation in neurodegenerative diseases and biopharmaceutical protein aggregation during production in mammalian cells. Despite obvious benefits, non-invasive quantitative assays for protein misfolding and mammalian cell aggregation have not been extensively exploited. In order to develop a simple non-invasive assay for protein misfolding and aggregation in mammalian cells, the folding reporter green fluorescent protein (GFP) system, originally developed for bacterial cells, was evaluated. As a folding reporter, GFP was fused to the C-terminus of a panel of human copper/zinc superoxide dismutase (SOD1) mutants with varying misfolding/aggregation propensities. Flow cytometric analysis of transfected HEK293T and NSC-34 cells revealed that the mean fluorescence intensities of the cells expressing GFP fusion of SOD1 variants exhibit an inverse correlation with the misfolding/aggregation propensities of the four SOD1 variants. These results support the hypothesis that the extent of misfolding/aggregation of a target protein in mammalian cells can be quantitatively estimated by measuring the mean fluorescence intensity of the cells expressing GFP fusion. The assay method developed here will pave the way for investigating protein misfolding/aggregation in mammalian cells.
Language
English
Published
University of Virginia, Department of Chemical Engineering, MS (Master of Science), 2012
Published Date
2012-03-29
Degree
MS (Master of Science)
Collection
Libra ETD Repository
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