Intein-catalyzed protein splicing: functional versatility and utility

内含肽催化的蛋白质剪接：功能多样性和实用性

• 批准号: RGPIN-2020-06083
• 负责人: Liu, Paul
• 金额: $ 2.33万
• 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: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=741017
• 关键词: Intein; catalyzed; protein; splicing; functional

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.

这项建议继续了我们对内含素催化的蛋白质剪接及其效用的长期研究。内含子在自然界中被发现是一类新型的酶蛋白，它催化蛋白质剪接反应来切除内含子，并用肽键连接两侧的宿主蛋白。我们最近专注于可以催化反式剪接反应来连接两个独立蛋白质的分裂内含子。Inteins通常可以作用于感兴趣的非天然蛋白质，并在蛋白质研究、蛋白质工程和生物技术的各个领域有许多用途。在过去的20年里，我的团队发现了大量的天然内含子，对内含子的进化和结构功能提出了见解，并设计出了用于蛋白质剪接和切割的有用内含子。在过去的六年里，我们在内含素催化的蛋白质和/或多肽连接工程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先进的分子生物学方法，包括定向进化，进一步开发内含素催化的蛋白质和/或肽连接的功能多样性(效率和通用性)。蛋白质和多肽的连接在蛋白质研究和工业的各个领域都是非常可取的。我们提出的研究是最接近于成功设计内含素衍生的蛋白质剪接连接酶的，这种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定点添加，这有望为包括药物蛋白质药物在内的许多与蛋白质相关的研究和工业领域提供巨大的价值。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特定部位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而嵌合的蜘蛛丝蛋白形成的各种人造蜘蛛丝。人造蜘蛛丝由于具有极强的强度、生物相容性和可生物降解性，已被广泛认为是潜在的有用生物材料。来自这一提议的人造蜘蛛丝可以使加拿大的研究人员和生物技术产业受益，例如，这些新型生物材料可以用于制造组织工程的支架。