Intein-catalyzed protein splicing: functional versatility and utility

这一建议是我们对内含肽催化蛋白质剪接及其应用的长期研究的继续。内含肽在自然界中被发现是一类新型的酶蛋白，其催化蛋白质剪接反应以切除内含肽并将侧翼宿主蛋白质与肽键连接。我们最近专注于分裂内含肽，可以催化反式剪接反应连接两个独立的蛋白质。内含肽通常可以在感兴趣的非天然蛋白质中起作用，并且在蛋白质研究、蛋白质工程和生物技术的各个领域中具有许多用途。在过去的20年里，我的团队发现了许多天然内含肽，产生了对内含肽进化和结构-功能的见解，并设计了有用的蛋白质剪接和切割内含肽。在过去的六年中，我们在工程内含肽催化的蛋白质和/或肽连接方面取得了重要突破。我们成功地将蛋白质连接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，进一步开发内含肽催化的蛋白质和/或肽连接的功能多样性（效率和通用性）。蛋白质和肽连接在蛋白质研究和工业的各个领域中是高度期望的。我们提出的研究是最接近于实现内含肽衍生的蛋白质剪接连接酶的成功工程化的，该蛋白质剪接连接酶通常可用于连接蛋白质和/或合成肽。这有望开辟生产化学合成蛋白质和半合成蛋白质的非常有用的途径，允许对蛋白质进行各种所需的化学修饰和非天然氨基酸的位点特异性添加，这有望为许多蛋白质相关研究和工业领域提供巨大价值，包括药物蛋白质药物。这些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位点特异性化学修饰或非天然氨基酸。 我们的第二个目标是将蛋白质连接应用于大量新型人工蜘蛛丝的生产，通过串联和组合蛋白质连接。这将使我们能够系统地探索由各种形式的大型嵌合蜘蛛丝蛋白形成的各种人造蜘蛛丝。人造蜘蛛丝由于具有强度高、生物相容性好、可生物降解等优点，被广泛认为是一种具有潜在应用价值的生物材料。这项建议的人造蜘蛛丝可以使加拿大研究人员和生物技术产业受益，例如，这些新的生物材料可以用于制造组织工程支架。

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.