Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells
Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells
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DOI：
10.1093/nar/gkf589
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发表时间：
2002-11-01
影响因子：
14.9
通讯作者：
Yokoyama, S
中科院分区：
文献类型：
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作者：
Sakamoto, K;Hayashi, A;Yokoyama, S
开发了抑制性tRNA（Tyr）和突变型酪氨酰-tRNA合成酶（TyrRS）对，以将3-碘-L-酪氨酸掺入哺乳动物细胞中的蛋白质中。首先，在D臂的三个位置突变大肠杆菌抑制tRNA（Tyr）基因，以产生用于表达的内部启动子。然而，这种tRNA与同源TyrRS一起未能在哺乳动物细胞中表现出抑制活性。然后，我们发现，琥珀抑制可以发生在异源对大肠杆菌TyrRS和嗜热脂肪芽孢杆菌抑制tRNA（Tyr），其天然含有启动子序列。此外，当抑制tRNA从基因簇表达时，这种抑制的效率显著提高，其中tRNA基因在相同方向上串联重复9次。为了掺入3-碘-L-酪氨酸，我们最近创建的其特异性大肠杆菌TyrRS变体TyrRS（V37 C195）与嗜热脂肪芽孢杆菌抑制tRNA（Tyr）一起在哺乳动物细胞中表达，而在生长培养基中提供3-碘-L-酪氨酸。因此，3-碘-L-酪氨酸以> 95%的占有率掺入蛋白质的琥珀位置。最后，我们证明了有条件的3-碘-L-酪氨酸掺入，从四环素调控的启动子的TyrRS（V37 C195）基因的诱导表达调节。
A suppressor tRNA(Tyr) and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNA(Tyr) gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNA(Tyr), which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNA(Tyr), while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter.