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: 6621676
• 负责人: JONATHAN BECKWITH
• 金额: $ 44.73万
• 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/6621676
• 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.

该建议旨在产生对蛋白质折叠，二硫键形成过程的重要特征的新见解，并提供生物技术工具，以增强多硫化物蛋白的产生。 I.我们将通过以下方式研究大肠杆菌周期中二硫键键异构化的过程：a）我们将确定从细胞质到毛囊的电子途径，以确保在降低状态下维持二硫键异构酶的维持； b）我们将确定二硫键异构酶，dsbc和dsbg的特异性以及其还原dsbd所需的蛋白质的特异性； c）我们将使用诱变和基因争吵改变这些蛋白质的特异性和活性，以了解其作用机理并增强异源二硫键键合蛋白的产生。 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期中多硫化物蛋白的产生。 ii。我们还将研究硫醇菌株的二硫键形成和异构化的过程，其硫醇改变：二硫化物氧化还原环境。 从缺少两个主要硫醇的菌株开始：二硫键氧化还原途径，我们具有孤立的抑制突变，可恢复对细胞质的足够还原能力以供生长，但仍允许蛋白质二硫键的细胞质形成。 这些抑制剂的研究已经揭示了大量细胞质氧化还原蛋白之间的新型相互作用。 我们将确定改变的基因和抑制剂突变会产生矛盾的还原性但氧化细胞质的机制。 我们将分析抑制剂的二硫键形成和异构化的动力学以及在异源二硫键蛋白的生产中的效率。因此，该提案将有关硫醇电子转移过程的广泛基础研究统一：氧化还原反应可能对医学上有用的产物的生物技术生产可能有益。