Creating a Selective Nanobody Against 3-Nitrotyrosine Containing Proteins.
Creating a Selective Nanobody Against 3-Nitrotyrosine Containing Proteins.
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DOI：
10.3389/fchem.2022.835229
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发表时间：
2022
影响因子：
5.5
通讯作者：
Mehl RA
中科院分区：
文献类型：
--
作者：
Van Fossen EM;Grutzius S;Ruby CE;Mourich DV;Cebra C;Bracha S;Karplus PA;Cooley RB;Mehl RA
开发氧化应激相关病理学的治疗方法的关键步骤是能够确定哪些特定的修饰蛋白质种类是病理学的无害副产物，哪些是病原体。为了实现这一目标，需要能够识别、表征和量化氧化翻译后修饰（oxPTM）的技术。纳米抗体（Nbs）由于其小尺寸、稳定性和可工程性而代表了用于细胞内分子追踪的精致工具。在这里，我们证明，这是可能的，以开发一个选择性的Nb对oxPTM蛋白质，与关键的进步是使用遗传密码扩展（GCE），以提供一个有效的来源，大量的高品质，同质和位点特异性oxPTM的Nb选择过程所需的蛋白质。在这项概念验证研究中，我们产生了一种对14-3-3信号蛋白的3-硝基酪氨酸（nitroTyr）修饰形式具有选择性的Nb，并且在其他蛋白质环境中对nitroTyr的识别较少。这一进展为GCE促进的其他抗PTM Nbs的开发打开了大门。
A critical step in developing therapeutics for oxidative stress-related pathologies is the ability to determine which specific modified protein species are innocuous by-products of pathology and which are causative agents. To achieve this goal, technologies are needed that can identify, characterize and quantify oxidative post translational modifications (oxPTMs). Nanobodies (Nbs) represent exquisite tools for intracellular tracking of molecules due to their small size, stability and engineerability. Here, we demonstrate that it is possible to develop a selective Nb against an oxPTM protein, with the key advance being the use of genetic code expansion (GCE) to provide an efficient source of the large quantities of high-quality, homogenous and site-specific oxPTM-containing protein needed for the Nb selection process. In this proof-of-concept study, we produce a Nb selective for a 3-nitrotyrosine (nitroTyr) modified form of the 14-3-3 signaling protein with a lesser recognition of nitroTyr in other protein contexts. This advance opens the door to the GCE-facilitated development of other anti-PTM Nbs.