| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2003: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2002: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
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.
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