A large variety of fusion tags have been developed to improve protein expression, solubilization, and purification. Nevertheless, these tags have been combined in a rather limited number of composite tags and usually these composite tags have been dictated by traditional commercially‐available expression vectors. Moreover, most commercially‐available expression vectors include either N‐ or C‐terminal fusion tags but not both. Here, we introduce TSGIT, a fusion‐tag system composed of both N‐ and a C‐terminal composite fusion tags. The system includes two affinity tags, two solubilization tags and two cleavable tags distributed at both termini of the protein of interest. Therefore, the N‐ and the C‐terminal composite fusion tags in TSGIT are fully orthogonal in terms of both affinity selection and cleavage. For using TSGIT, we streamlined the cloning, expression, and purification procedures. Each component tag is selected to maximize its benefits toward the final construct. By expressing and partially purifying the protein of interest between the components of the TSGIT fusion, the full‐length protein is selected over truncated forms, which has been a long‐standing problem in protein purification. Moreover, due to the nature of the cleavable tags in TSGIT, the protein of interest is obtained in its native form without any additional undesired N‐ or C‐terminal amino acids. Finally, the resulting purified protein is ready for efficient ligation with other proteins or peptides for downstream applications. We demonstrate the use of this system by purifying a large amount of native fluorescent mRuby3 protein and bacteriophage T7 gp2.5 ssDNA‐binding protein.

中文翻译：

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TSGIT：用于纯化天然蛋白和内含肽介导的连接就绪蛋白的 N 端和 C 端串联标签系统


已经开发了多种融合标签来改善蛋白质表达、溶解和纯化。然而，这些标签已被组合成数量相当有限的复合标签，并且通常这些复合标签是由传统的市售表达载体决定的。此外，大多数市售表达载体包含 N 端或 C 端融合标签，但不能同时包含两者。在这里，我们介绍 TSGIT，一个由 N 端和 C 端复合融合标签组成的融合标签系统。该系统包括分布在目标蛋白质两个末端的两个亲和标签、两个增溶标签和两个可切割标签。因此，TSGIT 中的 N 端和 C 端复合融合标签在亲和力选择和切割方面是完全正交的。为了使用 TSGIT，我们简化了克隆、表达和纯化程序。每个组件标签的选择都是为了最大限度地发挥其对最终构建的好处。通过表达和部分纯化 TSGIT 融合蛋白之间的目的蛋白，可以选择全长蛋白而不是截短形式，这一直是蛋白纯化中长期存在的问题。此外，由于 TSGIT 中可切割标签的性质，目标蛋白以其天然形式获得，没有任何额外的不需要的 N 或 C 末端氨基酸。最后，所得的纯化蛋白质可与其他蛋白质或肽有效连接以用于下游应用。我们通过纯化大量天然荧光 mRuby3 蛋白和噬菌体 T7 gp2.5 ssDNA 结合蛋白来演示该系统的用途。