Genetic encoding of unnatural amino acids for labeling proteins.
Genetic encoding of unnatural amino acids for labeling proteins.
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DOI：
10.1007/978-1-4939-2272-7_15
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发表时间：
2015-01-01
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影响因子：
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通讯作者：
Chin, Jason W
中科院分区：
文献类型：
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作者：
Lang, Kathrin;Davis, Lloyd;Chin, Jason W
通过遗传密码扩展的生物正交基团的位点特异性掺入为用任何探针位点特异性标记蛋白质提供了强有力的通用策略。在这里，我们描述了遗传编码的亲二烯体轴承非天然氨基酸到蛋白质中表达的大肠杆菌和哺乳动物细胞使用吡咯赖氨酰-tRNA合成酶/tRNACUA对及其变体。我们描述了快速荧光标记的蛋白质含有这些非天然氨基酸在体外，在E。大肠杆菌，并在活的哺乳动物细胞与四嗪荧光团共轭物在生物正交狄尔斯-阿尔德反应与反电子需求。这些方法已经扩展到动物中的位点特异性蛋白质标记，我们预计它们将对标记和成像领域产生广泛的影响。
The site-specific incorporation of bioorthogonal groups via genetic code expansion provides a powerful general strategy for site-specifically labeling proteins with any probe. Here we describe the genetic encoding of dienophile-bearing unnatural amino acids into proteins expressed in Escherichia coli and mammalian cells using the pyrrolysyl-tRNA synthetase/tRNACUA pair and its variants. We describe the rapid fluorogenic labeling of proteins containing these unnatural amino acids in vitro, in E. coli, and in live mammalian cells with tetrazine-fluorophore conjugates in a bioorthogonal Diels-Alder reaction with inverse electron demand. These approaches have been extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on the labeling and imaging field.