Novel screening protocol for multi-SS bond proteins using SEP tags and its application to the development of a minimal Luciferase

生物发光蛋白是生物成像不可或缺的工具，控制和操纵其分子特性已成为蛋白质工程的一个重要问题。在这里，我们开发了一种新的筛选方法，以提高生物发光特性的高斯荧光素酶（GLuc）。GLuc具有形成5个SS-键的10个半胱氨酸，这使得GLuc操作特别困难，因为当用传统方案在大肠杆菌中表达GLuc时，半胱氨酸变得缠结到非天然SS-键中。在本研究中，我们使用SEP标签来溶解蛋白质，并有利于在大肠杆菌中形成天然SS-键，大肠杆菌是一种方便且廉价的表达宿主。使用这种新技术，我们分离出具有红移发光的新型GLuc变体。该方法的应用范围可以扩展到其他含SS键的蛋白质，改进的GLuc将有助于补充报告蛋白的注册。

Bioluminescent proteins are indispensable tools for bio-imaging, and controlling and manipulating their molecular properties has become an important issue in protein engineering. Here we developed a novel screening method for improving the bioluminescent properties of Gaussia luciferase (GLuc). GLuc possesses 10 cysteins forming 5 SS-bonds, which make GLuc manipulation especially difficult, because the cysteins become entangled into non-native SS-bonds when GLuc is expressed in E-coli with traditional protocols. In the present study we used SEP tags to solubilize the protein and favor the formation of native SS-bonds in E-coli, which is a handy and inexpensive expression host. Using this new technique we isolated novel GLuc variants with red-shifted luminescence. The application range of this method can be extended to other SS-bond containing proteins, and the improved GLuc will contribute to complement the register of reporter proteins.