Protein photo-cross-tin king in mammalian cells by site-specific incorporation of a photoreactive amino acid
Protein photo-cross-tin king in mammalian cells by site-specific incorporation of a photoreactive amino acid
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DOI：
10.1038/nmeth739
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发表时间：
2005-03-01
期刊：
影响因子：
48
通讯作者：
Yokoyama, S
中科院分区：
文献类型：
--
作者：
Hino, N;Okazaki, Y;Yokoyama, S
我们报道了一种在哺乳动物细胞中光交联蛋白的方法，该方法基于通过使用扩展的遗传密码，位点特异性掺入光反应性氨基酸对苯甲酰基-L-苯丙氨酸（pBpa）。为了分析涉及衔接蛋白Grb 2的细胞信号传导相互作用，将pBpa掺入其Src同源2（SH 2）结构域中。将具有琥珀密码子的人GRB 2基因与嗜热脂肪芽孢杆菌抑制基因tRNA（Tyr）和大肠杆菌酪氨酰-tRNA合成酶（TyrRS）的pBpa特异性变体基因一起导入中国仓鼠卵巢（CHO）细胞。当pBpa包含在生长培养基中时，合成在琥珀位置具有pBpa的Grb 2变体。在暴露于365 nm的细胞紧密，蛋白质变体含有pBpa在近端的配体结合口袋的位置与瞬时表达的表皮生长因子（EGF）受体在EGF刺激的存在下交联。还检测到与内源性蛋白质的交联复合物。在体内光交联与pBpa纳入蛋白质将是有用的研究在哺乳动物细胞中的蛋白质-蛋白质相互作用。
We report a method of photo-cross-tin king proteins in mammalian cells, which is based on site-specific incorporation of a photoreactive amino acid, p-benzoyl-L-phenylalanine (pBpa), through the use of an expanded genetic code. To analyze the cell signaling interactions involving the adaptor protein Grb2, pBpa was incorporated in its Src homology 2 (SH2) domain. The human GRB2 gene with an amber codon was introduced into Chinese hamster ovary (CHO) cells, together with the genes for the Bacillus stearothermophilus suppressor tRNA(Tyr) and a pBpa-specific variant of Escherichia coli tyrosyl-tRNA synthetase (TyrRS). The Grb2 variant with pBpa in the amber position was synthesized when pBpa was included in the growth medium. Upon exposure of cells to 365-nm tight, protein variants containing pBpa in the positions proximal to the ligand-binding pocket were cross-linked with the transiently expressed epidermal growth factor (EGF) receptor in the presence of an EGF stimulus. Cross-linked complexes with endogenous proteins were also detected. In vivo photo-cross-linking with pBpa incorporated in proteins will be useful for studying protein-protein interactions in mammalian cells.