Genetic encoding of non-natural amino acids in Drosophila melanogaster Schneider 2 cells
Genetic encoding of non-natural amino acids in Drosophila melanogaster Schneider 2 cells
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DOI：
10.1002/pro.322
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发表时间：
2010-03-01
期刊：
影响因子：
8
通讯作者：
Yokoyama, Shigeyuki
中科院分区：
文献类型：
--
作者：
Mukai, Takahito;Wakiyama, Motoaki;Yokoyama, Shigeyuki
昆虫细胞可用于高产量生产重组蛋白，包括趋化因子和膜蛋白。在这项研究中，我们开发了一种基于昆虫细胞的系统，用于在特定位点将非天然氨基酸整合到蛋白质中。构建了三种类型的启动子系统，并比较了它们在果蝇Schneider 2细胞中表达原核琥珀抑制基因tRNA（Tyr）的效率。当与对3-碘-L-酪氨酸特异的大肠杆菌酪氨酰-tRNA合成酶的变体配对时，从U6启动子转录的抑制tRNA最有效地将氨基酸掺入细胞中的蛋白质中。然后在含有非天然氨基酸的蛋白质的产量方面比较了将这些原核分子瞬时和稳定引入昆虫细胞的方法，并且“瞬时”方法产生的产量高出7倍。通过这种方法，4-叠氮基-L-苯丙氨酸被掺入到人白细胞介素-8的特定位点。含叠氮基的IL-8的产量为1 μ g/1 mL细胞培养物，并且通过Staudinger-Bertozzi反应成功地用荧光探针标记了重组蛋白。
Insect cells are useful for the high-yield production of recombinant proteins including chemokines and membrane proteins. In this study, we developed an insect cell-based system for incorporating non-natural amino acids into proteins at specific sites. Three types of promoter systems were constructed, and their efficiencies were compared for the expression of the prokaryotic amber suppressor tRNA(Tyr) in Drosophila melanogaster Schneider 2 cells. When paired with a variant of Escherichia coli tyrosyl-tRNA synthetase specific for 3-iodo-L-tyrosine, the suppressor tRNA transcribed from the U6 promoter most efficiently incorporated the amino acid into proteins in the cells. The transient and stable introductions of these prokaryotic molecules into the insect cells were then compared in terms of the yield of proteins containing non-natural amino acids, and the "transient" method generated a sevenfold higher yield. By this method, 4-azido-L-phenylalanine was incorporated into human interleukin-8 at a specific site. The yield of the azido-containing IL-8 was 1 mu g/1 mL cell culture, and the recombinant protein was successfully labeled with a fluorescent probe by the Staudinger-Bertozzi reaction.