Dept. of Biological Sciences, KAIST, 373-1, Kusong-Dong, Yusong-Gu, Daejon 305-701, Korea.
Biotechnol Prog. 2010 Jan-Feb;26(1):246-51. doi: 10.1002/btpr.323.
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 hexahistidine-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 MS/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.
鉴定重组 CHO（rCHO）细胞中与未完全折叠和展开的促红细胞生成素（EPO）相互作用的细胞蛋白，有助于深入了解可能的遗传操作方法，以提高 EPO 的产量。为此，我们使用双标签（N 端 GST 和 C 端六组氨酸标签）EPO 在大肠杆菌中表达作为诱饵蛋白，rCHO 细胞（DG44）的细胞裂解物作为猎物蛋白，进行下拉实验。然后通过二维凝胶电泳（2DE）分离与双标签 EPO 相互作用的细胞蛋白，并通过 MALDI-TOF MS/MS 进行鉴定。成功鉴定出包括葡萄糖调节蛋白 78（GRP78）在内的总共 27 个蛋白点。GRP78 的 Western blot 分析证实了 MS 分析的结果。综上所述，下拉实验和蛋白质组学方法是鉴定 rCHO 细胞中外源蛋白相互作用的细胞蛋白的有效手段。
