Light Activation of Protein Splicing with a Photocaged Fast Intein
Light Activation of Protein Splicing with a Photocaged Fast Intein
复制标题
DOI：
10.1021/ja508597d
复制
发表时间：
2015-02-18
影响因子：
15
通讯作者：
Ai, Hui-wang
中科院分区：
文献类型：
--
作者：
Ren, Wei;Ji, Ao;Ai, Hui-wang
内含肽介导的蛋白质剪接在生物技术中有着广泛的应用。在此，我们描述了我们最近的结果，在工程的光活化内含肽与活的哺乳动物细胞兼容。将光笼化的半胱氨酸氨基酸残基遗传地引入高效的念珠藻（Npu）DnaE内含肽中。将所得的光笼内含肽插入红色荧光蛋白（RFP）mCherry和人Src酪氨酸激酶中以产生无活性的嵌合蛋白。光诱导的光化学反应能够重新激活内含肽并触发蛋白质剪接。如通过直接荧光成像或哺乳动物细胞中Src激酶传感器的成像所观察到的，形成了活性mCherry和Src。基因编码的光笼内含肽是一种通用的光遗传学工具，可以有效地光控制蛋白质的一级结构和功能
Intein-mediated protein splicing has found broad biotechnological applications. Herein, we describe our recent result in engineering a photoactivatable intein compatible with living mammalian cells. A photocaged cysteine amino acid residue was genetically introduced into a highly efficient Nostoc punctiforme (Npu) DnaE intein. The resulting photocaged intein was inserted into a red fluorescent protein (RFP) mCherry and a human Src tyrosine kinase to create inactive chimeric proteins. A light-induced photochemical reaction was able to reactivate the intein and trigger protein splicing. Active mCherry and Src were formed as observed by direct fluorescence imaging or imaging of an Src kinase sensor in mammalian cells. The genetically encoded photocaged intein is a general optogenetic tool, allowing effective photocontrol of primary structures and functions of proteins