Inteins: Expanding Biological Roles and Biotechnological Applications

项目总结/ABSTACT 这项应用的长期目标是更好地理解内含子作为后遗症的生物学重要性。 翻译调控元件，它们在病原体应激反应中的新角色，以及驾驭这种力量 为生物医学研究界发明有用的技术。Inteins，或 中间蛋白质是通过蛋白质从宿主基因中移除的自我催化的、可移动的遗传元件。 拼接。从应用的角度来看，内含素以高度特异的方式洗牌多肽键的能力 在蛋白质工程中被证明是特别有用的，导致了许多技术的发展。 而内含素的应用主导了他们的研究，并开发了基于内含素的新技术 通常，人们对自然界中内含子的生物学重要性知之甚少。Inteins具有丰富的移动性 微生物世界中的遗传元素，在大约一半的古菌和四分之一的古菌中发现 细菌。与长期以来认为内含子是分子寄生虫的假设相反，最近有越来越多的证据表明 这表明一些含有内含素的蛋白质已经进化成连接剪接，从而激活宿主蛋白， 对环境信号的影响。这代表了一种新颖的、可能广泛存在的后翻译形式 监管。此外，给定的内含子在人类中是不存在的，并且位于几个 病原体，了解调节蛋白质剪接的环境因素为可能的抗菌药提供信息 发展。 我们提出了以下两个目标，建立在最近的发现基础上，以及扩展到新的领域。 目标1将首次研究内含子作为新生链条所扮演的角色，广泛地确定 在从核糖体释放之前，剪接是可能的，也可以检查可能的应激反应。 细菌和真菌病原体的策略。AIM 2试图开发两种基于内含子的新技术。 第一，改善细菌中易错折叠蛋白表达的一般策略；第二，锌- 用于细菌和哺乳动物系统的受控自去除蛋白质纯化标签。穿过 这些目标，这项工作将增进我们对这些令人兴奋和未被研究的元素的作用的理解 在自然界中发挥作用，它们在病原体应激反应中的作用，并将导致基于蛋白质内含素的新技术 工程学。

PROJECT SUMMARY/ABSTACT The long-term goals of this application are to better understand the biological importance of inteins as post- translational regulatory elements, their emerging role in pathogen stress response, and to harness the power of their unique chemistry to invent useful technologies for the biomedical research community. Inteins, or intervening proteins, are self-catalytic, mobile genetic elements removed from host genes through protein splicing. From the applied perspective, the ability of inteins to shuffle peptide bonds in highly specific ways has proven exceptionally useful in protein engineering, leading to the development of numerous technologies. While intein applications have dominated their investigation, and new intein-based technologies are developed frequently, the biological importance of inteins in nature is poorly understood. Inteins are abundant mobile genetic elements in the microbial world, found in approximately one-half of archaea and one-quarter of bacteria. Contrary to long-standing assumption that inteins are molecular parasites, mounting recent evidence suggests that some intein-containing proteins have evolved to couple splicing, and thus host protein activation, to environmental signals. This represents a novel and potentially widespread form of post-translational regulation. Further, given inteins are absent in humans and located within essential genes of several pathogens, understanding the environmental factors that regulate protein splicing inform possible antimicrobial development. We propose the following two aims, building upon recent discoveries, as well as expanding into new arenas. Aim 1 will for the first time investigate the role inteins play as nascent chains, broadly determining whether splicing is possible prior to release from the ribosome, as well as examining possible stress response strategies of bacterial and fungal pathogens. Aim 2 seeks to develop two novel intein-based technologies. First, a general strategy to improve expression of misfolding-prone proteins in bacteria and second, a zinc- controlled self-removing protein purification tag for use in both bacterial and mammalian systems. Through these Aims, this work will enhance our understanding of the roles these exciting and understudied elements play in nature, their role in pathogen stress response, and will lead to new intein-based technologies for protein engineering.