Genetic engineering of proteins with cell membrane permeability
Genetic engineering of proteins with cell membrane permeability
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DOI：
10.1038/nbt0498-370
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发表时间：
1998-04-01
影响因子：
46.9
通讯作者：
Lin, YZ
中科院分区：
文献类型：
--
作者：
Rojas, M;Donahue, JP;Lin, YZ
产生具有固有细胞膜转运活性的蛋白质的方法的发现将扩大我们研究和操纵生命系统中各种细胞内过程的能力。我们报道了一种设计具有细胞膜通透性的蛋白质的方法。将12个氨基酸残基的膜转位序列融合到谷胱甘肽S转移酶的C端，得到的融合蛋白可有效地导入NIH3T3成纤维细胞和其他细胞。为了探索这种非破坏性的导入方法在细胞内过程研究中的适用性，我们测试了含有Grb2 SH2结构域的41 kDa GST-Grb2SH2-MTS融合蛋白对表皮生长因子(EGF)刺激的信号通路的影响。这种融合蛋白进入细胞，与磷酸化的EGF受体(EGFR)形成复合体，并抑制EGF诱导的EGFR-Grb2结合和丝裂原激活的蛋白激酶激活。
The discovery of methods for generating proteins with inherent cell membrane-translocating activity will expand our ability to study and manipulate various intracellular processes in living systems. We report a method to engineer proteins with cell-membrane permeability. After a 12-amino acid residue membrane-translocating sequence (MTS) was fused to the C-terminus of glutathione S-transferase (GST), the resultant GST-MTS fusion proteins were efficiently imported into NIH 3T3 fibroblasts and other cells. To explore the applicability of this nondestructive import method to the study of intracellular processes, a 41-kDa GST-Grb2SH2-MTS fusion protein containing the Grb2 SH2 domain was tested for its effect on the epidermal growth factor (EGF)-stimulated signaling pathway. This fusion protein entered cells, formed a complex with phosphorylated EGF receptor (EGFR), and inhibited EGF-induced EGFR-Grb2 association and mitogen-activated protein kinase activation.