Live Cell Imaging of Bioorthogonally Labelled Proteins Generated With a Single Pyrrolysine tRNA Gene.
Live Cell Imaging of Bioorthogonally Labelled Proteins Generated With a Single Pyrrolysine tRNA Gene.
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DOI：
10.1038/s41598-018-32824-1
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发表时间：
2018-09-28
影响因子：
4.6
通讯作者：
Arbely E
中科院分区：
文献类型：
--
作者：
Aloush N;Schvartz T;König AI;Cohen S;Brozgol E;Tam B;Nachmias D;Ben-David O;Garini Y;Elia N;Arbely E
遗传密码扩增使得非典型氨基酸（ncAA）能够掺入表达的蛋白质中。ncAA通常由终止密码子编码，该终止密码子由外源正交氨酰tRNA合成酶及其同源抑制tRNA（如吡咯赖氨酸对）解码。在这样的系统中，终止密码子抑制依赖于外源tRNA的细胞内水平。因此，编码多个拷贝的tRNAPyl基因（PylT）以改善ncAA掺入。然而，哺乳动物细胞中的某些应用，例如活细胞成像应用，其中标记的tRNA有助于背景荧光，可以从使用侵入性较小的最小表达系统中受益。因此，我们研究了tRNAPyl对生物正交标记的细胞内蛋白质的活细胞荧光成像的影响。我们发现，在C0S7细胞中，PylT拷贝数的减少对蛋白质表达水平没有可测量的影响。重要的是，减少PylT拷贝数通过增强信噪比和减少不动的tRNAPyl群体来改善活细胞图像的质量。这使我们能够改善生物正交标记的细胞内蛋白质的活细胞成像，并同时标记细胞中的两种不同蛋白质。我们的研究结果表明，根据转染的细胞系，掺入ncAA，和应用，引入的PylT基因的数量可以最小化。
Genetic code expansion enables the incorporation of non-canonical amino acids (ncAAs) into expressed proteins. ncAAs are usually encoded by a stop codon that is decoded by an exogenous orthogonal aminoacyl tRNA synthetase and its cognate suppressor tRNA, such as the pyrrolysine pair. In such systems, stop codon suppression is dependent on the intracellular levels of the exogenous tRNA. Therefore, multiple copies of the tRNAPyl gene (PylT) are encoded to improve ncAA incorporation. However, certain applications in mammalian cells, such as live-cell imaging applications, where labelled tRNAs contribute to background fluorescence, can benefit from the use of less invasive minimal expression systems. Accordingly, we studied the effect of tRNAPyl on live-cell fluorescence imaging of bioorthogonally-labelled intracellular proteins. We found that in COS7 cells, a decrease in PylT copy numbers had no measurable effect on protein expression levels. Importantly, reducing PylT copy numbers improved the quality of live-cell images by enhancing the signal-to-noise ratio and reducing an immobile tRNAPyl population. This enabled us to improve live cell imaging of bioorthogonally labelled intracellular proteins, and to simultaneously label two different proteins in a cell. Our results indicate that the number of introduced PylT genes can be minimized according to the transfected cell line, incorporated ncAA, and application.
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影响因子：
16.6
作者：
Chen, Peng R.;Groff, Dan;Guo, Jiantao;Ou, Weijia;Cellitti, Susan;Geierstanger, Bernhard H.;Schultz, Peter G.
通讯作者：
Schultz, Peter G.
影响因子：
15
作者：
Blackman, Melissa L.;Royzen, Maksim;Fox, Joseph M.
通讯作者：
Fox, Joseph M.
影响因子：
48
作者：
Elsässer SJ;Ernst RJ;Walker OS;Chin JW
通讯作者：
Chin JW
影响因子：
16.6
作者：
Coin, Irene;Perrin, Marilyn H.;Vale, Wylie W.;Wang, Lei
通讯作者：
Wang, Lei
DOI：
10.1073/pnas.1309584110
发表时间：
2013-07-16
影响因子：
11.1
作者：
Chatterjee, Abhishek;Xiao, Han;Schultz, Peter G.
通讯作者：
Schultz, Peter G.