Intein-catalyzed protein splicing: functional versatility and utility

这一建议继续了我们对内部催化的蛋白质剪接及其应用的长期研究。肠内蛋白是自然界中发现的一类新型酶促蛋白，它催化蛋白质剪接反应，去除肠内蛋白，并用肽键连接两侧的宿主蛋白。我们最近专注于分裂蛋白，它可以催化反式剪接反应，连接两个独立的蛋白质。蛋白质通常可以作用于非天然蛋白质，在蛋白质研究、蛋白质工程和生物技术的各个领域都有许多用途。在过去的20年里，我的团队发现了许多天然的内部蛋白，对内部蛋白的进化和结构功能产生了见解，并设计了有用的蛋白质剪接和切割内部蛋白。在过去的六年里，我们在工程内部催化的蛋白质和/或肽连接方面取得了重要突破。我们成功地将蛋白结扎技术应用于人造蜘蛛丝的生产和分析。在接下来的五年里，我们的第一个目标是利用包括定向进化在内的先进分子生物学方法，进一步开发内部催化蛋白质和/或肽连接的功能多样性（效率和普遍性）。蛋白质和肽的连接在蛋白质研究和工业的各个领域都是非常需要的。我们所提出的研究是最接近于成功地实现蛋白衍生剪接连接酶的工程设计，该酶通常可用于连接蛋白质和/或合成肽。这有望为生产化学合成蛋白质和半合成蛋白质开辟非常有用的途径，允许在蛋白质上添加各种所需的化学修饰和非天然氨基酸，这有望为许多蛋白质相关的研究和工业领域提供巨大的价值，包括药用蛋白质药物。这可以使许多加拿大研究人员和生物技术公司受益，他们经常希望在他们感兴趣的蛋白质中添加特定位点的化学修饰或非天然氨基酸。我们的第二个目标是通过串联和组合蛋白质连接，将蛋白质连接应用于大量新型人造蜘蛛丝的生产。这将使我们能够系统地探索由各种形式的大型嵌合蛛丝蛋白形成的各种人造蛛丝。人造蜘蛛丝被广泛认为是一种潜在的有用的生物材料，因为它们非常坚固，具有生物相容性和可生物降解性。该提案中的人造蜘蛛丝可以使加拿大的研究人员和生物技术行业受益，例如，这些新型生物材料可以用于制造组织工程的支架。

This proposal continues our long-term study of intein-catalyzed protein splicing and its utilities. Inteins are found in nature as a novel class of enzymatic proteins that catalyzes a protein splicing reaction to excise the intein and ligate the flanking host proteins with a peptide bond. We recently focused on split-inteins that can catalyze a trans-splicing reaction to ligate two separate proteins. Inteins can generally work in non-native proteins of interest and have many uses in various fields of protein research, protein engineering and biotechnology. In past 20 years, my group has discovered numerous natural inteins, produced insights on intein evolution and structure-function, and engineered useful inteins for protein splicing and cleavage. In the last six years, we have made important breakthroughs in engineering intein-catalyzed protein and/or peptide ligations. We successfully used protein ligation in production and analysis of artificial spider silks. Over the next five years, our first goal is to further develop the functional versatility (efficiency and generality) of intein-catalyzed protein and/or peptide ligations, using advanced molecular biology methods including directed evolution. Protein and peptide ligations are highly desirable in various fields of protein research and industry. Our proposed research is the closest to achieving a successful engineering of intein-derived protein splicing ligase that can be generally useful for ligating proteins and/or synthetic peptides. This is expected to open up tremendously useful avenues of producing chemically synthesized proteins and semi-synthetic proteins, allowing site-specific additions of various desired chemical modifications and unnatural amino acids to proteins, which are expected to provide great values to many protein-related research and industrial fields including pharmaceutical protein drugs. These can benefit many Canadian researchers and biotech companies who often wish to add site-specific chemical modifications or unnatural amino acids to their proteins of interest. Our second goal is to apply protein ligations to the production of a large number of novel artificial spider silks, through tandem and combinatorial protein ligations. This will allow us to explore systematically various artificial spider silks formed from various forms of large and chimeric spider silk proteins. Artificial spider silks have been widely recognized as potentially useful biomaterials, because they are exceptionally strong, biocompatible, and biodegradable. Artificial spider silks from this proposal can benefit Canadian researchers and biotech industry, for example, these novel biomaterials may be used in making scaffoldings for tissue engineering.