Cloning, Purification And Crystallization Of The Subunit Of The Electron Transfer Complex AC Ⅲ In Thermophilic Photosynthetic Bacteria Roseiflexus

The filamentous anoxygenic phototrophic bacterium Roseiflexus possesses an unique electron transfer complex called Alternative Complex Ⅲ(AC Ⅲ) instead of conventional cytochrome bc1 type complex. ACIII behaves as a menaquinol:auracyanin oxidoreductase in the photosynthetic electron transfer chain. It is a multisubunit integral membrane protein complex. It is found in a variety of photosynthetic and nonphotosynthetic bacteria. Previous studies have shown that of subunit of ACⅢ named ActE is the key subunit of whole complex. However, there are some debet on its effect on the overall structure and function of ACⅢ. In order to enhance the understanding of the working mechanism of ACⅢ complex in photosynthetic electron transport chain, it is necessary to explore the structure and function of each subunit of ACⅢ.In this project, we utilized molecular biology and protein chemistry technology to study ActE subunit protein by carrying out the following experiments.(1) After modification of the growth condition, the large scale cultivation of Roseiflexus castenholzii was conducted. The yield of harvested cell increased in new condition.(2)By selecting the optimal detergent and solubilization condition of membrane components of ultracentrifuation, ACⅢ was extracted effectivedly and isolated through the methods of several chromatography steps.(3) By analysising of the genome of Roseiflexus castenholzii in JGI databank, the specific primers were designed to clone the actE gene by PCR. The PCR products of the target gene were ligated to new type of expression plasmid pEASY-E1. The recombinant vector was transformed into the expression host cell E.coli BL21(DE3).(4) By using the methods of affinity chromatography, ion exchange chromatography and gel filtration chromatography, the heterogleous expressed AcE sununit protein was succefully isolated and purified.(5) By using different kind of the protein crystallization kits, the crystallization conditions of the above purified ActE protein and ACⅢ complex were intensively searched. The promsing condtion was found out in the screening. In general, out results provide a theoretical basis for the further study of the structure and function of ACⅢ.