University of Chinese Academy of Sciences, National Center for Protein Science Shanghai, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 Yueyang Road, 200031, Shanghai, China.
Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland.
Commun Biol. 2020 Dec 10;3(1):753. doi: 10.1038/s42003-020-01478-z.
Green fluorescent proteins (GFPs) are widely used to monitor membrane protein expression, purification, and stability. An ideal reporter should be stable itself and provide high sensitivity and yield. Here, we demonstrate that a coral (Galaxea fascicularis) thermostable GFP (TGP) is by such reasons an improved tag compared to the conventional jellyfish GFPs. TGP faithfully reports membrane protein stability at temperatures near 90 °C (20-min heating). By contrast, the limit for the two popular GFPs is 64 °C and 74 °C. Replacing GFPs with TGP increases yield for all four test membrane proteins in four expression systems. To establish TGP as an affinity tag for membrane protein purification, several high-affinity synthetic nanobodies (sybodies), including a non-competing pair, are generated, and the crystal structure of one complex is solved. Given these advantages, we anticipate that TGP becomes a widely used tool for membrane protein structural studies.
绿色荧光蛋白（GFPs）被广泛用于监测膜蛋白的表达、纯化和稳定性。理想的报告蛋白应该自身稳定，并提供高灵敏度和产量。在这里，我们证明了一种珊瑚（Galaxea fascicularis）耐热 GFP（TGP）是一种比传统水母 GFP 更好的标签。TGP 忠实地报告了接近 90°C（加热 20 分钟）的温度下膜蛋白的稳定性。相比之下，两种流行的 GFP 的极限是 64°C 和 74°C。用 TGP 代替 GFP 可提高四个表达系统中所有四个测试膜蛋白的产量。为了将 TGP 确立为膜蛋白纯化的亲和标签，生成了几个高亲和力的合成纳米抗体（sybodies），包括一对非竞争的纳米抗体，并解决了其中一个复合物的晶体结构。鉴于这些优势，我们预计 TGP 将成为膜蛋白结构研究中广泛使用的工具。
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