Site-specific protein modification using lipoic acid ligase and bis-aryl hydrazone formation.
Site-specific protein modification using lipoic acid ligase and bis-aryl hydrazone formation.
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DOI：
10.1002/cbic.201100764
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发表时间：
2012-04-16
期刊：
影响因子：
--
通讯作者：
Ting AY
中科院分区：
文献类型：
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作者：
Cohen JD;Zou P;Ting AY
筛选大肠杆菌Trp 37突变体。大肠杆菌硫辛酸连接酶（LplA）产生能够将芳基-醛或芳基-肼底物连接到LplA的13-氨基酸受体肽（LPL）的酶。一旦位点特异性地连接到重组蛋白，芳基醛可以用肼-探针缀合物化学选择性地衍生化，并且芳基肼可以以类似的方式用偶联探针缀合物衍生化。AlexaFluor 568在体外靶向单价链霉亲和素，并靶向活哺乳动物细胞表面上的neurexin-1β，证明了这种两步标记。为了进一步强调这种技术，我们还用荧光藻红蛋白蛋白标记活细胞表面的低密度脂蛋白受体，以允许单分子成像和随时间的跟踪。
A screen of Trp37 mutants of E. coli lipoic acid ligase (LplA) produced enzymes capable of ligating an aryl-aldehyde or an aryl-hydrazine substrate to LplA’s 13-amino acid acceptor peptide (LAP). Once site-specifically attached to recombinant proteins, aryl-aldehydes could be chemo-selectively derivatized with hydrazine-probe conjugates, and aryl-hydrazines could be derivatized in an analogous manner with aldehyde–probe conjugates. Such two-step labeling was demonstrated for AlexaFluor568 targeting to monovalent streptavidin in vitro, and to neurexin-1β on the surface of living mammalian cells. To further highlight this technique, we also labeled low density lipoprotein receptor on the surface of live cells with fluorescent phycoerythrin protein to allow single molecule imaging and tracking over time.