A fluorophore ligase for site-specific protein labeling inside living cells
A fluorophore ligase for site-specific protein labeling inside living cells
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DOI：
10.1073/pnas.0914067107
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发表时间：
2010-06-15
影响因子：
11.1
通讯作者：
Ting, Alice Y.
中科院分区：
文献类型：
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作者：
Uttamapinant, Chayasith;White, Katharine A.;Ting, Alice Y.
生物显微镜将受益于绿色荧光蛋白的较小替代品，用于对活细胞中的特定蛋白质进行成像。在这里，我们介绍了一种在活细胞中进行多肽融合重组蛋白荧光标记的方法，这种方法具有很高的特异性。Prime使用的是一种工程化的荧光团连接酶，这种连接酶来自天然的大肠杆菌硫辛酸连接酶(LplA)。通过结构导向突变，我们创造了一种突变的连接酶，它能够识别7-羟基香豆素底物，并催化其共价结合成可转座的13个氨基酸的多肽，称为LAP(LplA受体多肽)。我们发现这种荧光团连接在细胞中发生在10分钟内，并且它对LAP融合蛋白具有高度的特异性，超过了所有内源性哺乳动物蛋白。通过将Prime连接酶基因定位于特定的亚细胞室，我们能够选择性地标记空间上不同的蛋白质亚集，如Neurein的表面池和肌动蛋白的核池。
Biological microscopy would benefit from smaller alternatives to green fluorescent protein for imaging specific proteins in living cells. Here we introduce PRIME (PRobe Incorporation Mediated by Enzymes), a method for fluorescent labeling of peptide-fused recombinant proteins in living cells with high specificity. PRIME uses an engineered fluorophore ligase, which is derived from the natural Escherichia coli enzyme lipoic acid ligase (LplA). Through structure-guided mutagenesis, we created a mutant ligase capable of recognizing a 7-hydroxycoumarin substrate and catalyzing its covalent conjugation to a transposable 13-amino acid peptide called LAP (LplA Acceptor Peptide). We showed that this fluorophore ligation occurs in cells in 10 min and that it is highly specific for LAP fusion proteins over all endogenous mammalian proteins. By genetically targeting the PRIME ligase to specific subcellular compartments, we were able to selectively label spatially distinct subsets of proteins, such as the surface pool of neurexin and the nuclear pool of actin.