Biocentre, Johannes-Gutenberg University Mainz, 55128, Mainz, Germany.
Institute of Molecular Biology gGmbH, 55128, Mainz, Germany.
Chembiochem. 2020 Nov 16;21(22):3216-3219. doi: 10.1002/cbic.202000338. Epub 2020 Jul 23.
Genetic code expansion (GCE) is a versatile tool to site-specifically incorporate a noncanonical amino acid (ncAA) into a protein, for example, to perform fluorescent labeling inside living cells. To this end, an orthogonal aminoacyl-tRNA-synthetase/tRNA (RS/tRNA) pair is used to insert the ncAA in response to an amber stop codon in the protein of interest. One of the drawbacks of this system is that, in order to achieve maximum efficiency, high levels of the orthogonal tRNA are required, and this could interfere with host cell functionality. To minimize the adverse effects on the host, we have developed an inducible GCE system that enables us to switch on tRNA or RS expression when needed. In particular, we tested different promotors in the context of the T-REx or Tet-On systems to control expression of the desired orthogonal tRNA and/or RS. We discuss our result with respect to the control of GCE components as well as efficiency. We found that only the T-REx system enables simultaneous control of tRNA and RS expression.
遗传密码扩展（GCE）是一种将非天然氨基酸（ncAA）特异性地掺入蛋白质中的多功能工具，例如，在活细胞内进行荧光标记。为此，使用正交氨酰-tRNA 合成酶/tRNA（RS/tRNA）对来响应感兴趣蛋白质中的琥珀终止密码子插入 ncAA。该系统的一个缺点是，为了实现最高效率，需要高水平的正交 tRNA，这可能会干扰宿主细胞功能。为了最大限度地减少对宿主的不利影响，我们开发了一种诱导型 GCE 系统，使我们能够在需要时打开 tRNA 或 RS 的表达。特别是，我们在 T-REx 或 Tet-On 系统的背景下测试了不同的启动子，以控制所需的正交 tRNA 和/或 RS 的表达。我们根据 GCE 组件的控制和效率讨论了我们的结果。我们发现只有 T-REx 系统能够同时控制 tRNA 和 RS 的表达。
