Taking down the FLAG! How Insect Cell Expression Challenges an Established Tag-System

In 1988 the preceding journal of Nature Biotechnology, Bio/Technology, reported a work by Hopp and co-workers about a new tag system for the identification and purification of recombinant proteins: the FLAG-tag. Beside the extensively used hexa-his tag system the FLAG-tag has gained broad popularity due to its small size, its high solubility, the presence of an internal Enterokinase cleavage site, and the commercial availability of high-affinity anti-FLAG antibodies. Surprisingly, considering the heavy use of FLAG in numerous laboratories world-wide, we identified in insect cells a post-translational modification (PTM) that abolishes the FLAG-anti-FLAG interaction rendering this tag system ineffectual for secreted proteins. The present publication shows that the tyrosine that is part of the crucial FLAG epitope DYK is highly susceptible to sulfation, a PTM catalysed by the enzyme family of Tyrosylprotein-Sulfo-transferases (TPSTs). We showed that this modification can result in less than 20% of secreted FLAG-tagged protein being accessible for purification questioning the universal applicability of this established tag system.

1988年，《自然-生物技术》（Nature Biotechnology）的前身期刊《Bio/Technology》刊载了霍普（Hopp）及其同事开发的一种用于重组蛋白鉴定与纯化的新型标签系统——FLAG标签（FLAG-tag）。除了应用广泛的六聚组氨酸标签（hexa-his tag）系统外，FLAG标签凭借其体积小巧、可溶性优异、自带肠激酶（Enterokinase）内部酶切位点，以及高亲和力抗FLAG抗体的商业化可得性，获得了广泛的普及。尽管FLAG标签在全球众多实验室中被大量使用，但令人意外的是，我们在昆虫细胞中发现了一种翻译后修饰（post-translational modification，PTM），该修饰会破坏FLAG与抗FLAG抗体的结合，使得该标签系统无法有效用于分泌型蛋白的纯化。本研究证实，构成FLAG关键表位DYK的酪氨酸极易发生硫酸化修饰，这类修饰由酪氨酰蛋白质磺基转移酶（Tyrosylprotein-Sulfo-transferases，TPSTs）家族催化。我们的研究显示，该修饰会导致可被纯化的分泌型FLAG标签融合蛋白占比不足20%，这对这一成熟标签系统的普适适用性提出了质疑。