Protein Chemical Modification Inside Living Cells Using Split Inteins.
Protein Chemical Modification Inside Living Cells Using Split Inteins.
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DOI：
10.1007/978-1-4939-6451-2_8
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Camarero, Julio A
中科院分区：
文献类型：
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作者：
Borra, Radhika;Camarero, Julio A
可视化、跟踪、测量、扰乱或激活活细胞中蛋白质的方法是表征和理解细胞内生命的空间和时间基础的生物医学工作的核心。尽管荧光蛋白已被证明对于蛋白质功能的体内研究非常有用，但它们的实用性本质上是有限的，因为它们的光谱和结构特征是相互依赖的。这些限制刺激了蛋白质化学标记替代方法的创建。我们在本协议中描述了使用荧光共振发射转移 (FRET) 淬灭的 DnaE 分裂内含子进行位点特异性标记和活细胞中蛋白质的伴随荧光激活。我们已经使用几种人类细胞系成功地采用这种方法对转录因子 YY1 的 DNA 结合域 (DBD) 进行位点特异性细胞内标记。此外，我们已经证明这种方法还可以用于修饰蛋白质，以控制它们的细胞定位并可能改变它们的生物活性。
Methods to visualize, track, measure, and perturb or activate proteins in living cells are central to biomedical efforts to characterize and understand the spatial and temporal underpinnings of life inside cells. Although fluorescent proteins have proven to be extremely useful for in vivo studies of protein function, their utility is inherently limited because their spectral and structural characteristics are interdependent. These limitations have spurred the creation of alternative approaches for the chemical labeling of proteins. We describe in this protocol the use of fluorescence resonance emission transfer (FRET)-quenched DnaE split-inteins for the site-specific labeling and concomitant fluorescence activation of proteins in living cells. We have successfully employed this approach for the site-specific in-cell labeling of the DNA binding domain (DBD) of the transcription factor YY1 using several human cell lines. Moreover, we have shown that this approach can be also used for modifying proteins in order to control their cellular localization and potentially alter their biological activity.