Inducible Genetic Code Expansion in Eukaryotes

被引：10
作者：
Koehler, Christine [1 ,2 ,3 ,4 ,5 ]
Girona, Gemma Estrada [3 ,4 ]
Reinkemeier, Christopher D. [1 ,2 ,3 ,4 ]
Lemke, Edward A. [1 ,2 ,3 ,4 ]
机构：
[1] Johannes Gutenberg Univ Mainz, Bioctr, D-55128 Mainz, Germany
[2] Inst Mol Biol gGmbH, D-55128 Mainz, Germany
[3] European Mol Biol Lab, Struct & Computat Biol Unit, Meyerhofstr 1, D-69117 Heidelberg, Germany
[4] European Mol Biol Lab, Cell Biol & Biophys Unit, Meyerhofstr 1, D-69117 Heidelberg, Germany
[5] ARAXA Biosci GmbH, Meyerhofstr 1, D-69117 Heidelberg, Germany
来源：
CHEMBIOCHEM | 2020年 / 21卷 / 22期
关键词：
amber suppression; PylRS; Tet-On; T-REx; unnatural amino acid; UNNATURAL AMINO-ACIDS; SUPPRESSOR TRANSFER-RNAS; MAMMALIAN-CELLS; EXPRESSION; SYSTEM; EVOLUTION; U6;
D O I：
10.1002/cbic.202000338
中图分类号：
Q5 [生物化学]; Q7 [分子生物学];
学科分类号：
071010 ; 081704 ;
摘要：
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.
引用
收藏
页码：3216 / 3219
页数：4
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