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首页> 外文期刊>Biotechnology Progress >A Proteomic Approach for Identifying Cellular Proteins Interacting with Erythropoietin in Recombinant Chinese Hamster Ovary Cells
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A Proteomic Approach for Identifying Cellular Proteins Interacting with Erythropoietin in Recombinant Chinese Hamster Ovary Cells

机译：蛋白质组学方法在重组中国仓鼠卵巢细胞中鉴定与促红细胞生成素相互作用的细胞蛋白

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摘要

Identification of the cellular proteins interacting with incompletely folded and unfolded forms of erythropoietin (EPO) in recombinant CHO (rCHO) cells leads to better insight into the possible genetic manipulation approaches for increasing EPO production. To do so, a pull-down assay was performed with dual-tagged (N-terminal GST- and C-terminal hexahis-tidine-tagged) EPO expressed in E. coli as bait proteins and cell lysates of rCHO cells (DG44) as prey proteins. Cellular proteins interacting with dual-tagged EPO were then resolved by two-dimensional gel electrophoresis (2DE) and identified by MALDI-TOF MSI MS. A total of 27 protein spots including glucose-regulated protein 78 (GRP78) were successfully identified. Western blot analysis of GRP78 confirmed the results of the MS analyses. Taken together, a pull-down assay followed by a proteomic approach is found to be an efficient means to identify cellular proteins interacting with foreign protein in rCHO cells.
机译：鉴定与重组CHO（rCHO）细胞中不完全折叠和未折叠形式的促红细胞生成素（EPO）相互作用的细胞蛋白，可以更好地了解增加EPO产量的可能的遗传操作方法。为此，使用在大肠杆菌中表达为诱饵蛋白的双标签（N端GST和C端六巯基吡啶标签的）EPO作为诱饵蛋白和rCHO细胞的细胞裂解液（DG44）作为猎物蛋白。然后通过二维凝胶电泳（2DE）解析与双标签EPO相互作用的细胞蛋白，并通过MALDI-TOF MSI MS进行鉴定。总共成功鉴定出27个蛋白点，包括葡萄糖调节蛋白78（GRP78）。 GRP78的蛋白质印迹分析证实了MS分析的结果。综上所述，在蛋白质组学方法中进行下拉测定是发现在rCHO细胞中鉴定与外源蛋白质相互作用的细胞蛋白质的有效方法。

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