Xu Zhen, Rui Yan-Ning, Balzeau Julien, Menezes Miriam R, Niu Airu, Hagan John P, Kim Dong H
Department of Neurosurgery, The University of Texas Health Science Center at Houston, TX, United States.
Department of Neurosurgery, The University of Texas Health Science Center at Houston, TX, United States.
Biochem Biophys Res Commun. 2017 Aug 12;490(1):8-16. doi: 10.1016/j.bbrc.2017.05.153. Epub 2017 May 30.
Protein tagging with a wide variety of epitopes and/or fusion partners is used routinely to dissect protein function molecularly. Frequently, the required DNA subcloning is inefficient, especially in cases where multiple constructs are desired for a given protein with unique tags. Additionally, the generated clones have unwanted junction sequences introduced. To add versatile tags into the extracellular domain of the transmembrane protein THSD1, we developed a protein tagging technique that utilizes non-classical type IIS restriction enzymes that recognize non-palindromic DNA sequences and cleave outside of their recognition sites. Our results demonstrate that this method is highly efficient and can precisely fuse any tag into any position of a protein in a scarless manner. Moreover, this method is cost-efficient and adaptable because it uses commercially available type IIS restriction enzymes and is compatible with the traditional cloning system used by many labs. Therefore, precision tagging technology will benefit a number of researchers by providing an alternate method to integrate an array of tags into protein expression constructs.
使用多种表位和/或融合伴侣进行蛋白质标记常用于从分子层面剖析蛋白质功能。通常，所需的DNA亚克隆效率低下，尤其是在需要为给定蛋白质构建多个带有独特标签的构建体的情况下。此外，生成的克隆会引入不需要的连接序列。为了将通用标签添加到跨膜蛋白THSD1的胞外结构域，我们开发了一种蛋白质标记技术，该技术利用非经典IIS型限制酶，这些酶识别非回文DNA序列并在其识别位点之外切割。我们的结果表明，该方法效率极高，能够以无痕方式将任何标签精确融合到蛋白质的任何位置。此外，该方法具有成本效益且适应性强，因为它使用市售的IIS型限制酶，并且与许多实验室使用的传统克隆系统兼容。因此，精确标记技术将为众多研究人员提供一种替代方法，将一系列标签整合到蛋白质表达构建体中，从而使他们受益。
