Second-generation covalent TMP-tag for live cell imaging.
Second-generation covalent TMP-tag for live cell imaging.
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DOI：
10.1021/ja303374p
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发表时间：
2012-08-22
影响因子：
15
通讯作者：
Cornish VW
中科院分区：
文献类型：
--
作者：
Chen Z;Jing C;Gallagher SS;Sheetz MP;Cornish VW
化学标签现在是荧光蛋白的可行替代品，用于用具有改善的亮度和其他专门性质的有机荧光团标记活细胞中的蛋白质。最近，我们成功地使我们的TMP-标签共价与蛋白质标签和配体荧光标记之间的邻近诱导反应。然而，这种最初的设计受到缓慢的体外标记动力学和有限的活细胞蛋白标记的影响。因此，在这里，我们报告了第二代共价TMP标签，具有快速标记半衰期，可以很容易地标记活细胞中的各种细胞内蛋白质。具体而言，我们设计了一种丙烯酰胺-甲氧苄啶-荧光团（A-TMP-荧光团v2.0）亲电体，其具有优化的接头，用于与大肠杆菌二氢叶酸还原酶（eDHFR）的TMP结合口袋外工程化的半胱氨酸（Cys）亲核试剂快速反应，并开发了一种有效的化学合成方法，用于常规生产各种A-TMP-探针v2.0标签。然后，我们筛选了一组eDHFR：Cys变体，并鉴定eDHFR：L28 C在体外与A-TMP-生物素v2.0反应的半衰期为8分钟。最后，我们证明了用A-TMP-荧光素、A-TMP-Dapoxyl和A-TMP-Atto 655对各种细胞蛋白质靶标的活细胞成像。凭借其稳健性，这种第二代共价TMP标签增加了有限数量的化学标签，可用于有效地共价标记活细胞中的细胞内蛋白质。此外，这种第二代设计的成功进一步验证了邻近诱导反应性和有机化学不仅是化学标签工程的工具，而且更广泛地用于合成生物学。
Chemical tags are now viable alternatives to fluorescent proteins for labeling proteins in living cells with organic fluorophores that have improved brightness and other specialized properties. Recently, we successfully rendered our TMP-tag covalent with a proximity-induced reaction between the protein tag and the ligand-fluorophore label. This initial design, however, suffered from slow in vitro labeling kinetics and limited live cell protein labeling. Thus, here we report a second-generation covalent TMP-tag that has a fast labeling half-life and can readily label a variety of intracellular proteins in living cells. Specifically, we designed an acrylamide-trimethoprim-fluorophore (A-TMP-fluorophore v2.0) electrophile with an optimized linker for fast reaction with a cysteine (Cys) nucleophile engineered just outside the TMP-binding pocket of Escherichia coli dihydrofolate reductase (eDHFR) and developed an efficient chemical synthesis for routine production of a variety of A-TMP-probe v2.0 labels. We then screened a panel of eDHFR:Cys variants and identified eDHFR:L28C as having an 8-min half-life for reaction with A-TMP-biotin v2.0 in vitro. Finally, we demonstrated live cell imaging of various cellular protein targets with A-TMP-fluorescein, A-TMP-Dapoxyl, and A-TMP-Atto655. With its robustness, this second-generation covalent TMP-tag adds to the limited number of chemical tags that can be used to covalently label intracellular proteins efficiently in living cells. Moreover, the success of this second-generation design further validates proximity-induced reactivity and organic chemistry as tools not only for chemical tag engineering but also more broadly for synthetic biology.
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DOI：
10.1126/science.1198830
发表时间：
2011-03-11
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Hoskins AA;Friedman LJ;Gallagher SS;Crawford DJ;Anderson EG;Wombacher R;Ramirez N;Cornish VW;Gelles J;Moore MJ
通讯作者：
Moore MJ
影响因子：
3.2
作者：
Krusemark, Casey J.;Belshaw, Peter J.
通讯作者：
Belshaw, Peter J.
影响因子：
15
作者：
Izumi, Saki;Urano, Yasuteru;Nagano, Tetsuo
通讯作者：
Nagano, Tetsuo
影响因子：
2.9
作者：
BACCANARI, DP;DALUGE, S;KING, RW
通讯作者：
KING, RW
影响因子：
15
作者：
BRESLOW, R;CORCORAN, RJ;KALEYA, R
通讯作者：
KALEYA, R