Inteins: Expanding Biological Roles and Biotechnological Applications

项目概要/摘要 本申请的长期目标是更好地理解内含肽作为后蛋白的生物学重要性。 翻译调控元件，它们在病原体应激反应中的新作用，并利用这种能力， 他们独特的化学性质，为生物医学研究界发明有用的技术。内含肽，或 间插蛋白是自催化的、移动的遗传元件，通过蛋白质从宿主基因中移除 拼接从应用的角度来看，内含肽以高度特异性的方式改组肽键的能力， 被证明在蛋白质工程中非常有用，导致了许多技术的发展。 内含肽的应用已成为研究的主要方向，新的内含肽技术也在不断发展 内含肽在自然界中的生物学重要性常常知之甚少。内含肽是丰富的移动的 微生物世界中的遗传元素，在大约一半的古生菌和四分之一的 细菌与长期以来认为内含肽是分子寄生虫的假设相反，最近越来越多的证据表明， 提示一些内含肽蛋白质已经进化为偶联剪接，从而激活宿主蛋白质， 环境信号。这代表了一种新的和潜在的广泛的形式的翻译后 调控此外，给定的内含肽在人类中不存在，并且位于几种哺乳动物的必需基因内。 病原体，了解调节蛋白质剪接的环境因素， 发展 我们提出以下两个目标，建立在最近的发现，以及扩展到新的领域。 Aim 1将首次研究内含肽作为新生链发挥的作用，广泛地确定是否 在从核糖体释放之前，剪接是可能的，以及检查可能的应激反应 细菌和真菌病原体的策略。目标2旨在开发两种基于内含肽的新技术。 首先，一个通用的策略，以提高表达的错误折叠倾向的蛋白质在细菌和第二，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。通过 这些目标，这项工作将提高我们对这些令人兴奋的和未充分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，并将导致新的蛋白质内含肽技术 工程.

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.