Evolutionary and functional versatility of inteins

本研究的目的是研究一种名为肠蛋白的新型蛋白质元件。肠内蛋白催化一种称为蛋白质剪接的独特反应，这是一种蛋白质成熟事件，将肠内蛋白从前体蛋白中移除，形成成熟的功能蛋白。在NSERC之前的资助下，我们已经发现了各种形式和大小的天然意图，设计了具有各种理想功能的人工意图，并建立了一个在实验室条件下改善意图功能的定向进化系统。我们建议继续进行这些联合研究，以实现以下目标。1)通过内嵌蛋白片段互补构建可调控的内嵌蛋白，实现高效的蛋白剪接，作为调控其他感兴趣蛋白功能的分子开关。2)将各种需要的蛋白质标记物或标签插入到内含物中，构建功能性的内含物标记盒，这可以作为重组蛋白生产和纯化的通用工具。3)通过合理设计和定向进化，设计出能够将合成肽添加到重组蛋白上的专用蛋白，并利用这些蛋白开发位点特异性蛋白标记和修饰的新方法，为蛋白质研究和生产提供有价值的工具。4)通过专门设计的定向进化，生产出既快速又通用的超级肠道，这可以极大地扩展基于肠道的技术的通用效用。由于蛋白质在生命科学研究和生物技术中具有重要的地位，因此这些建议的研究具有学术和现实意义。

The objective of this proposed research is to study a novel protein element named intein. Inteins catalyze a unique reaction termed protein splicing, which is a protein maturation event that removes the intein from a precursor protein to form the mature and functional protein. Supported by a previous NSERC grant, we have discovered natural inteins of various forms and sizes, engineered artificial inteins capable of various desirable functions, and established a directed evolution system for improving intein functions under laboratory conditions. We propose to continue these intein studies to achieve the following goals. 1) To construct controllable inteins capable of efficient protein splicing through intein fragment complementation, which may be useful as a molecular switches to control the functions of other proteins of interest. 2) To construct functional intein-marker cassettes by inserting various desirable protein markers or tags into inteins, which can be generally useful tools for the production and purification of recombinant proteins. 3) To engineer specialty inteins capable of adding synthetic peptides onto recombinant proteins, through rational design and directed evolution, and use these inteins to develop novel methods of site-specific protein labeling and modifications, which can be valuable tools of protein research and production. 4) To produce super inteins that are both fast and general, through specially designed directed evolution, which can greatly expand the general utilities of intein-based technologies. These proposed researches have both academic and practical significances, because proteins are of central importance in life science research and in biotechnology.