Single-Site Labeling of Native Proteins Enabled by a Chemoselective and Site-Selective Chemical Technology
Single-Site Labeling of Native Proteins Enabled by a Chemoselective and Site-Selective Chemical Technology
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DOI：
10.1021/jacs.8b10490
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发表时间：
2018-11-07
影响因子：
15
通讯作者：
Rai, Vishal
中科院分区：
文献类型：
--
作者：
Adusumalli, Srinivasa Rao;Rawale, Tdattatraya Gautam;Rai, Vishal
化学生物学研究通常需要将标记精确地共价连接到天然蛋白质上。人们寻求这种方法来探测、设计和调节蛋白质的性质。目前，由于缺乏授权化学技术，这一需求在很大程度上没有得到满足。在这里，我们报告了一个化学平台，使天然蛋白质的位点选择性标记。最初，可逆的分子间反应将“化学关键”全面地置于所有可接近的赖氨酸残基上。这些关键蛋白具有驱动蛋白质的位点选择性共价标记的能力。关键在生理条件下分离，并使各种标签的后期安装获得能力。化学平台是模块化的，并且试剂设计调节修饰位点。关键是一个多任务组，有利于纯化标记的蛋白质，消除了额外的色谱标签的要求。该方法允许标记蛋白质混合物中的单一蛋白质。对蛋白质中一个可接近的残基进行精确的修饰，可以确保它们的结构保持不变。标记后肌红蛋白、细胞色素C、醛缩酶和溶菌酶C的酶活性保持保守。此外，修饰的胰岛素的细胞摄取及其下游信号传导过程保持不受干扰。关键销定向修饰（LDM）为荧光团和药物与Fab和单克隆抗体的缀合提供了方便的途径。其递送对Her-2阳性SKBR-3乳腺癌细胞具有选择性抗增殖活性的曲妥珠单抗-多柔比星和曲妥珠单抗-美坦辛缀合物。
Chemical biology research often requires precise covalent attachment of labels to the native proteins. Such methods are sought after to probe, design, and regulate the properties of proteins. At present, this demand is largely unmet due to the lack of empowering chemical technology. Here, we report a chemical platform that enables site-selective labeling of native proteins. Initially, a reversible intermolecular reaction places the "chemical linchpins" globally on all the accessible Lys residues. These linchpins have the capability to drive site-selective covalent labeling of proteins. The linchpin detaches within physiological conditions and capacitates the late-stage installation of various tags. The chemical platform is modular, and the reagent design regulates the site of modification. The linchpin is a multitasking group and facilitates purification of the labeled protein eliminating the requirement of additional chromatography tag. The methodology allows the labeling of a single protein in a mixture of proteins. The precise modification of an accessible residue in protein ensures that their structure remains unaltered. The enzymatic activity of myoglobin, cytochrome C, aldolase, and lysozyme C remains conserved after labeling. Also, the cellular uptake of modified insulin and its downstream signaling process remain unperturbed. The linchpin directed modification (LDM) provides a convenient route for the conjugation of a fluorophore and drug to a Fab and monoclonal antibody. It delivers trastuzumab-doxorubicin and trastuzumab-emtansine conjugates with selective antiproliferative activity toward Her-2 positive SKBR-3 breast cancer cells.