Live Cell Imaging of Bioorthogonally Labelled Proteins Generated With a Single Pyrrolysine tRNA Gene

被引：23

作者：

Aloush, Noa ^{[1
,3
]}

Schvartz, Tomer ^{[2
,3
]}

Konig, Andres I. ^{[2
,3
]}

Cohen, Sarit ^{[1
,3
]}

Brozgol, Eugene ^{[4
,5
]}

Tam, Benjamin ^{[1
]}

Nachmias, Dikla ^{[2
,3
]}

Ben-David, Oshrit ^{[1
,3
]}

Garini, Yuval ^{[4
,5
]}

Elia, Natalie ^{[2
,3
]}

Arbely, Eyal ^{[1
,2
,3
]}

机构：

[1] Ben Gurion Univ Negev, Dept Chem, POB 653, IL-8410501 Beer Sheva, Israel

[2] Ben Gurion Univ Negev, Dept Life Sci, POB 653, IL-8410501 Beer Sheva, Israel

[3] Ben Gurion Univ Negev, Natl Inst Biotechnol Negev, POB 653, IL-8410501 Beer Sheva, Israel

[4] Bar Ilan Univ, Phys Dept, IL-5290002 Ramat Gan, Israel

[5] Bar Ilan Univ, Inst Nanotechnol, IL-5290002 Ramat Gan, Israel

来源：

SCIENTIFIC REPORTS | 2018年 / 8卷

基金：

以色列科学基金会; 欧洲研究理事会;

关键词：

UNNATURAL AMINO-ACIDS; SITE-SPECIFIC INCORPORATION; MAMMALIAN-CELLS; CODE EXPANSION; MUTAGENESIS; SYSTEM; CHEMISTRY; LIGATION; DELIVERY; PLASMID;

D O I：

10.1038/s41598-018-32824-1

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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 synthetase/tRNA(CUA)(Pyl) pair. In such systems, stop codon suppression is dependent on the intracellular levels of the exogenous tRNA. Therefore, multiple copies of the tRNA(Pyl) 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 tRNA(Pyl) 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 tRNA(Pyl) 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.

引用

页数：11

共 48 条

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