Evolutionary and functional versatility of inteins

内含子的进化和功能多样性

• 批准号: 41823-2010
• 负责人: Liu, Paul
• 金额: $ 4.08万
• 依托单位: Dalhousie University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2011
• 资助国家: 加拿大
• 起止时间: 2011-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=474902
• 关键词: Evolutionary; functional; versatility; inteins

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.

本研究的目的是研究一种名为内含肽的新型蛋白质元件。 内含肽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，将内含肽从前体蛋白中去除以形成成熟和功能性蛋白质。 在NSERC的资助下，我们发现了各种形式和大小的天然内含肽，工程化的人工内含肽能够实现各种期望的功能，并建立了一个定向进化系统，用于在实验室条件下改善内含肽的功能。 我们建议继续这些内含肽的研究，以实现以下目标。 1)构建能够通过内含肽片段互补进行有效蛋白质剪接的可控内含肽，其可能用作控制其他感兴趣蛋白质功能的分子开关。 2)通过将各种所需的蛋白质标记或标签插入内含肽中来构建功能性内含肽标记盒，其通常可以是用于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，设计出能够将合成肽添加到重组蛋白上的特异性内含肽，并利用这些内含肽开发新的蛋白质位点特异性标记和修饰方法，这将成为蛋白质研究和生产的有价值的工具。 4)通过专门设计的定向进化，产生既快速又通用的超级内含肽，这可以极大地扩展内含肽技术的通用性。 蛋白质在生命科学和生物技术中具有重要的地位，因此这些研究具有重要的学术意义和实际意义。