A revisited folding reporter for quantitative assay of protein misfolding and aggregation in mammalian cells.
A revisited folding reporter for quantitative assay of protein misfolding and aggregation in mammalian cells.
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DOI：
10.1002/biot.201200103
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发表时间：
2012-10
影响因子：
4.7
通讯作者：
Kwon, Inchan
中科院分区：
文献类型：
--
作者：
Gregoire, Simpson;Kwon, Inchan
蛋白质的错误折叠和聚集在许多生理过程中起着重要的作用。这些包括神经退行性疾病中的病理性蛋白质聚集和哺乳动物细胞中生产期间的生物药物蛋白质聚集。为了开发一种简单的非侵入性测定哺乳动物细胞中的蛋白质错误折叠和聚集，折叠报告绿色荧光蛋白（GFP）系统，最初开发的细菌细胞，进行了评估。作为一个折叠的报告，GFP融合到一组人铜/锌超氧化物歧化酶（SOD 1）突变体的C-末端，具有不同的错误折叠/聚集倾向。对转染的HEK 293 T和NSC-34细胞的流式细胞术分析显示，表达SOD 1变体的GFP融合体的细胞的平均荧光强度与四种SOD 1变体的错误折叠/聚集倾向呈负相关。我们的研究结果支持的假设，在哺乳动物细胞中的靶蛋白的错误折叠/聚集的程度可以定量估计通过测量平均荧光强度的细胞表达GFP融合。本研究所建立的检测方法将有助于理解SOD 1变异体的聚集过程和识别聚集抑制剂。该方法也有很大的希望在哺乳动物细胞中的其他蛋白质的错误折叠/聚集研究。
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. 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. 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 here will facilitate understanding of aggregation process of SOD1 variants and identifying aggregation inhibitors. The method also has great promise for misfolding/ aggregation study of other proteins in mammalian cells.
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DOI：
10.1016/j.molbrainres.2005.07.010
发表时间：
2005-10-31
期刊：
MOLECULAR BRAIN RESEARCH
影响因子：
--
作者：
Babetto, E;Mangolini, A;Cantoni, L
通讯作者：
Cantoni, L
影响因子：
12.4
作者：
Jang, Jae-Hyung;Koerber, James T.;Schaffer, David V.
通讯作者：
Schaffer, David V.
影响因子：
4.8
作者：
Ghanta, J;Shen, CL;Murphy, RM
通讯作者：
Murphy, RM
影响因子：
2.9
作者：
Masuda, Masami;Suzuki, Nobuyuki;Hasegawa, Masato
通讯作者：
Hasegawa, Masato
影响因子：
3.7
作者：
GRAHAM, FL;VANDEREB, AJ
通讯作者：
VANDEREB, AJ