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.

该建议旨在对蛋白质折叠的重要特征二硫键形成过程产生新的见解，并为增强多二硫蛋白的生产提供生物技术工具。1 .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途径，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的特异性，以及它们还原所需的蛋白质DsbD的特异性；c)我们将使用诱变和基因混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提高异源二硫结合蛋白的产生；d)我们将描述新基因，这些基因在多拷贝或突变改变时，增强了外质中多二硫蛋白的产生。2。我们还将研究在改变硫醇二硫氧化还原环境的大肠杆菌菌株细胞质中二硫键形成和异构化的过程。从缺少两种主要硫醇二硫氧化还原途径的菌株开始，我们分离出抑制突变，这些突变恢复了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和机制，从而抑制突变产生矛盾的还原但氧化细胞质。我们将分析抑制因子对二硫键形成和异构化的动力学以及对异源二硫键结合蛋白的生产效率的影响。因此，该建议将硫醇氧化还原反应中电子转移过程的广泛基础研究与可能对医学有用产品的生物技术生产有益的研究结合起来。