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Similarity and Diversity of Electron Transport Pathways to Nitrogense, Studies on Rhodobacter and Mesorhizobium

氮电子传输途径的相似性和多样性，红细菌和中生根瘤菌的研究

基本信息

批准号：
14540594
负责人：
SAEKI Kazuhiko
金额：
$ 2.18万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-14540594/
关键词：
RHODOBACTER FERREDOXIN ELECTRON-TRANSPORT NITROGEN-FIXATION NADH MEMBANE-PROTEIN-COMPLEX

项目摘要

For structural and biochemical analysis of proteins that participate electron transport to nitrogenase, the following studies were carried out on in Rhodobacter capsulatus rnf and nif gene products and Mesorhizobium loti fixABCX products. (1) Affinity chromatography procedures were optimized for successful purification of the proteins under strict anaerobic conditions with dithionite-containing buffer system, using (His)6-tagged model electron carrier protein. (2) Effective purification procedures were established for R. capsulatus NifF protein after expression in Escherichia coli. (3) The M loti fixX gene was over-expressed in E. coli under various conditions including co-expression of GroES-GroEL chaperones, however, the products formed inclusion body. Reconstitution conditions were investigated to make holo-FixX with Fe-S cluster from denatured inclusion bodies. (4) The M loti fixA, fixB, fixC and fixX genes were co-expressed in E. coli, however, most of the product were situated on membranes. (5) For effective exploitation of the genome sequence information of Lotus microsymbiont, Mesorhizobiurn loti MAFF303099, to discover gene functions, we have constructed an ordered and mutually overlapping cosmid library using an IncP broad host range vector. The library consisted of 480 clones to cover approximately 99.6% of the genome with average insert size and overlap of 26.9 and 11.1 kbp, respectively.

为了对参与固氮酶电子传递的蛋白质进行结构和生化分析，对荚膜红杆菌rnf和nif基因产物以及中索根瘤菌fixABCX产物进行了以下研究。 (1) 优化亲和层析程序，使用 (His)6 标记的模型电子载体蛋白，在严格厌氧条件下使用含连二亚硫酸盐的缓冲系统成功纯化蛋白质。 (2)建立了荚膜红囊菌NifF蛋白在大肠杆菌中表达后的有效纯化方法。 (3)MlotifixX基因在大肠杆菌中在GroES-GroEL分子伴侣共表达等多种条件下过表达，但产物形成包涵体。研究了从变性包涵体制备具有 Fe-S 簇的 Holo-FixX 的重构条件。 (4)Mloti fixA、fixB、fixC和fixX基因在大肠杆菌中共表达，但大部分产物位于膜上。 (5)为了有效利用莲花微共生体Mesorhizobiurn loti MAFF303099的基因组序列信息来发现基因功能，我们使用IncP广宿主范围载体构建了有序且相互重叠的粘粒文库。该文库由 480 个克隆组成，覆盖约 99.6% 的基因组，平均插入片段大小和重叠分别为 26.9 和 11.1 kbp。