蛋白质是生命科学、生物工程、医学制药研发的主要对象，也是生物能源转化、工业生物催化等多个领域的重要创新源泉。但是，蛋白质的内在不稳定性限制了其表征与应用，也是导致蛋白质折叠异常、诱发疾病的重要原因之一。 本项目拟开发新型蛋白质体内稳定性检测探针，为利用定向进化技术优化蛋白质稳定性提供新的手段。大肠杆菌中尿卟啉原III甲基转移酶CysGA催化内源底物产生红色荧光，我们发现将其与不同目标蛋白融合表达时，其荧光表型与目标蛋白的稳定性呈正相关性。根据这一现象，我们拟筛选CysGA表面位点，将其进行拆分以打造“三明治”形式的插入型探针，进一步提升蛋白质稳定性筛选的效率。后续在两个方面考察CysGA探针系统的应用：1. 获取两种目标蛋白稳定性提升的突变体，以促进其结构与机制表征；2.筛选化学小分子库，获取抑制淀粉样纤维生成的新型小分子，为治疗2型糖尿病等蛋白质折叠疾病提供新的思路。

Proteins are the main research targets for biological science, bioengineering, pharmaceutical science, and so on. They are also an important source of innovation in many fields such as bioenergy conversion and industrial biocatalysis. However, proteins are intrinsically unstable, which limited their characterization and application, and is also one of the major causes of protein misfolding and aggregation. To provide a new means to stabilizing proteins with directed evolution techniques, we intend to develop a novel in vivo biosensor based on the uroporphyrinogen III methyltransferase, CysGA, from Escherichia coli. CysGA catalyzes red fluorescent substances formation in E.coli from endogenous substrates. We previously found that the fluorescence identity of the cells positively correlated with the stability of test proteins fused to CysGA, indicating the potential to adapt CysGA into a protein folding sensor. We will screen the surface exposed residues in CysGA to find the sites suitable for foreign protein insertion, making a sandwich fusion between the target protein and CysGA. Then, we will explore the application of this system in the following aspects: 1. Obtaining stabilized variants of two target proteins in order to promote their structural and mechanistic characterization; 2. Screening a small molecule library to find new types of inhibitors for hIAPP amyloid formation, which will provide new ideas for the treatment of type-2 diabetes and other protein folding diseases.