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

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，本研究对胶囊红杆菌rnf和nif基因产物和中生根瘤菌固定物ABCX产物进行了研究。(1)利用(His)6标记的模型电子载体蛋白，优化了亲和层析程序，在严格的厌氧条件下，使用含二亚硫酸盐的缓冲体系，成功地纯化了蛋白质。(2)建立了在大肠杆菌中表达的胶囊乳杆菌Niff蛋白的有效纯化方法。(3)Mloti Fixx基因在包括GroES-GroEL伴侣蛋白共表达在内的多种条件下均能在大肠杆菌中高效表达，但表达产物形成包涵体。研究了变性包涵体与铁-S簇包涵体制备全息固定的条件。(4)Mloti FixA、FixB、FixC和Fixx基因在大肠杆菌中共表达，但大部分产物定位于膜上。(5)为了有效利用莲花微共生菌MAFF303099的基因组序列信息，以发现其基因功能，我们利用IncP广宿主范围载体构建了一个有序且相互重叠的粘粒文库。文库由480个克隆组成，覆盖基因组的99.6%，平均插入大小为26.9kbp，重叠率为11.1kbp。

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.