Evolutionary and functional versatility of inteins

这项拟议研究的目的是研究一种名为内蛋白的新蛋白质元件。内含子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，它将内含子从前体蛋白中移除，形成成熟的功能蛋白质。在先前NSERC资助的支持下，我们发现了各种形式和大小的自然内含子，设计了具有各种理想功能的人工内含子，并建立了在实验室条件下改进内含子功能的定向进化系统。我们建议继续进行这些INTIN研究，以实现以下目标。1)构建能够通过内含子片段互补进行有效蛋白质剪接的可控内含子，这可能有助于作为分子开关来控制其他目的蛋白的功能。2)通过在内含子中插入各种理想的蛋白质标记或标签来构建功能性内含子标记盒，这通常是生产和纯化重组蛋白的有用工具。3)通过合理的设计和定向进化，设计出能够在重组蛋白上添加合成肽的特殊内含子，并利用这些内含子开发新的蛋白质定点标记和修饰方法，为蛋白质的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，产生既快速又通用的超级整数，极大地扩展了基于INTIN的技术的通用用途。这些研究具有重要的理论和实践意义，因为蛋白质在生命科学研究和生物技术中具有重要意义。

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.