title: A revisited folding reporter for quantitative assay of protein misfolding and aggregation in mammalian cells.
abstract: Protein misfolding and aggregation play important roles in many physiological processes. These include pathological protein aggregation in neurodegenerative diseases and biopharmaceutical protein aggregation during production in mammalian cells. 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 exhibited an inverse correlation with the misfolding/aggregation propensities of the four SOD1 variants. Our 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 herein will facilitate the understanding of aggregation process of SOD1 variants and the identification of aggregation inhibitors. The method also has great promise for misfolding/aggregation studies of other proteins in mammalian cells.
authors.affiliation: Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904, USA.
authors.forename: Simpson
authors.initials: S
authors.lastname: Gregoire
authors.forename: Inchan
authors.initials: I
authors.lastname: Kwon
mesh_terms: D001709:Biotechnology / Q000379:methods*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D011401:Promoter Regions, Genetic; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013482:Superoxide Dismutase