RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan.
Protein Sci. 2010 Mar;19(3):440-8. doi: 10.1002/pro.322.
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 microg/1 mL cell culture, and the recombinant protein was successfully labeled with a fluorescent probe by the Staudinger-Bertozzi reaction.
昆虫细胞可用于高效生产重组蛋白，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，可在特定位置将非天然氨基酸掺入蛋白质中。构建了三种启动子系统，并比较了它们在果蝇 Schneider 2 细胞中表达原核琥珀终止密码子 tRNA（Tyr）的效率。与对 3-碘-L-酪氨酸特异的大肠杆菌酪氨酸-tRNA 合成酶的变体配对时，转录自 U6 启动子的抑制 tRNA 最有效地将该氨基酸掺入细胞中的蛋白质中。然后，根据含有非天然氨基酸的蛋白质的产量比较了这些原核分子在昆虫细胞中的瞬时和稳定引入，“瞬时”方法产生了七倍的更高产量。通过该方法，将 4-叠氮基-L-苯丙氨酸掺入人白细胞介素-8 的特定位置。含叠氮基的 IL-8 的产量为 1μg/1mL 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。
