本项目以断裂蛋白内含子介导的蛋白质反式剪接为基础，拟开发出蛋白质定点标记和修饰的通用技术。在蛋白质研究领域，人们常常希望对蛋白质进行标记和修饰，然而目前缺乏位点特异性的有效标记方法。我们将利用前期工作中已获得的具有高剪接活性、相互间无交叉反应的多个S1型和S11型断裂蛋白内含子进行蛋白质位点特异性的双末端标记，如荧光素、聚乙二醇化修饰等。同时，我们拟构建Cysteine-free S1型和S11型断裂蛋白内含子，以减少其对目的蛋白的影响及降低成本，并对已有的断裂蛋白内含子进行体外定向进化，以期获得剪接效率更高、通用性更好的新型断裂蛋白内含子。该技术解决了传统的化学方法标记蛋白质的非特异性、产物不均一性等问题，同时定向进化突变体也将有助于深入了解蛋白内含子的结构与功能的关系。

We will study intein-based protein trans-splicing and focus on its applications in protein labeling and modification. Split intein has been used in protein ligation, cyclization,labeling and modifications,alough the lower or no splicing in heterogenous exteins, insoluble of recombinant fusion proteins limited its further applications. High splicing efficiency and more general inteins will be available through directed evolution of S1 and S11 split inteins.For practical uses, we tried to splice two different fluoresceins or biotins to the N and C terminus of recombinant model protein di-ubiquitin using S1 and S11 split-inteins site-specifically. PEGylation of protein or peptide medicines will also included.Through absorbent spectra,fluorescence spectra and MS analysis,we plan to establish a general site-specific protein labelling method. This will produce technologies of protein engineering that are useful for modifications of recombinant proteins.This will also advance our knowledge of intein structure-function and to explore the potential of intein.