ANALYSIS OF PROTEIN DISULFIDE BOND FORMATION IN E. COLI

大肠杆菌中蛋白质二硫键形成的分析

• 批准号: 6847392
• 负责人: JONATHAN BECKWITH
• 金额: $ 46.22万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6847392
• 关键词: Escherichia coli; NAD(P)H oxidoreductase; bacterial genetics; bacterial proteins; biotechnology; directed evolution; disulfide bond; electron transport; functional /structural genomics; gene mutation; isomer; isomerase; molecular cloning; molecular genetics; oxidation reduction reaction; protein disulfide reductase (glutathione); protein engineering; protein folding; recombinant proteins; thiols; thioredoxin

This proposal is designed to yield new insights into an important feature of protein folding, the process of disulfide bond formation, and to provide biotechnological tools for enhanced production of multidisulfide proteins. I. We will study the process of disulfide bond isomerization in the Escherichia coli periplasm in the following ways: a) We will determine the pathways of electrons from cytoplasm to periplasm that insure the maintenance of disulfide bond isomerases in the reduced state; b)We will determine the specificity of the disulfide bond isomerases, DsbC and DsbG, and of the protein necessary for their reduction, DsbD; c)We will alter the specificity and activity of these proteins using mutagenesis and gene scrambling in order both to understand the mechanism of their action and to enhance the production of heterologous disulfide-bonded proteins; d)We will characterize new genes, which in multi-copy or when altered by mutation, enhance the production of multi-disulfide proteins in the periplasm. II. We will also study the process of disulfide bond formation and isomerization in the cytoplasm of strains of E. coli with altered thiol:disulfide redox environments. Beginning with strains missing the two major thiol: disulfide redox pathways, we have isolated suppressor mutations that restore sufficient reductive power to the cytoplasm for growth, but still allow cytoplasmic formation of protein disulfide bonds. Studies of these suppressors are already revealing novel interactions among the large set of cytoplasmic redox proteins. We will determine the altered genes and the mechanisms whereby suppressor mutations generate the paradoxical reducing but oxidizing cytoplasms. We will analyze suppressor for the kinetics of disulfide bond formation and isomerization and for the efficiency in the production of heterologous disulfide bonded proteins. This proposal thus unites extensive basic studies on processes of electron transfer in thiol: redox reactions with likely benefits for the biotechnological production of medically useful products.

该提案旨在对蛋白质折叠的一个重要特征（二硫键形成过程）产生新的见解，并为增强多二硫键蛋白质的生产提供生物技术工具。 I. 我们将通过以下方式研究大肠杆菌周质中二硫键异构化的过程： a) 我们将确定电子从细胞质到周质的路径，以确保二硫键异构酶维持在还原状态； b) 我们将确定二硫键异构酶 DsbC 和 DsbG 以及还原它们所需的蛋白质 DsbD 的特异性； c)我们将通过诱变和基因扰乱来改变这些蛋白质的特异性和活性，以便了解它们的作用机制并增强异源二硫键蛋白质的生产； d）我们将表征新基因，这些基因在多拷贝或通过突变改变时，会增强周质中多二硫键蛋白的产生。 二.我们还将研究硫醇：二硫键氧化还原环境改变的大肠杆菌菌株细胞质中二硫键形成和异构化的过程。 从缺少两个主要硫醇：二硫键氧化还原途径的菌株开始，我们分离出抑制突变，这些突变可以恢复细胞质生长所需的足够还原能力，但仍然允许细胞质形成蛋白质二硫键。 对这些抑制因子的研究已经揭示了大量细胞质氧化还原蛋白之间的新相互作用。 我们将确定改变的基因以及抑制突变产生矛盾的还原性但氧化性细胞质的机制。 我们将分析抑制器的二硫键形成和异构化动力学以及异源二硫键蛋白的生产效率。因此，该提案将硫醇中电子转移过程的广泛基础研究结合起来：氧化还原反应可能有益于医用产品的生物技术生产。