Zhang Ziwei, Xu Huan, Si Longlong, Chen Yi, Zhang Bo, Wang Yan, Wu Yiming, Zhou Xueying, Zhang Lihe, Zhou Demin
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
Biochem Biophys Res Commun. 2017 Aug 5;489(4):490-496. doi: 10.1016/j.bbrc.2017.05.178. Epub 2017 May 31.
The genetic incorporation of unnatural amino acids (Uaas) with defined properties into proteins at designated sites represents an extremely powerful tool for protein engineering. However, the efficient incorporation of Uaas in response to the amber stop codon in mammalian cells remains a substantial challenge due to the competition from release factor 1(RF1). Addressing this challenge will greatly broaden the power and scope of this technology. Here, we chose the eRF1 mutant, which can selectively enhance Uaa incorporation in response to the amber codon without increasing the readthrough of the opal and ochre codons. Then, we developed an engineered stable cell line using a tetracycline-controlled inducible lentiviral system for the conditional expression of mutant eRF1, which can minimize the potential effect on normal translation termination. Using the eRF1-engineered cells, we provided a 2-fold improvement in the yield of protein containing a Uaa incorporated at a single site, with the protein yield approaching 90% of the wild-type control without the amber codon. Moreover, we achieved the successful incorporation of Uaas at four sites in various proteins at a measured level of 20%.
将具有特定性质的非天然氨基酸（Uaas）在指定位点遗传掺入蛋白质中是蛋白质工程中一项极其强大的工具。然而，由于释放因子1（RF1）的竞争，在哺乳动物细胞中响应琥珀色终止密码子高效掺入Uaas仍然是一项重大挑战。应对这一挑战将极大地扩展该技术的能力和范围。在此，我们选择了eRF1突变体，它可以选择性地增强对琥珀色密码子的Uaa掺入，而不会增加乳白和赭石密码子的通读。然后，我们使用四环素控制的诱导型慢病毒系统开发了一种工程稳定细胞系，用于条件性表达突变型eRF1，这可以将对正常翻译终止的潜在影响降至最低。使用eRF1工程细胞，我们将在单个位点掺入Uaa的蛋白质产量提高了2倍，蛋白质产量接近无琥珀色密码子的野生型对照的90%。此外，我们成功地在各种蛋白质的四个位点以20%的测量水平掺入了Uaas。
