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。

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.