The Purification Of The Core-membrane Linker And The Studies On Some Genes Function In Cyanobacteria

In this paper, the purification of the core-membrane linker from Anabaena sp. PCC7120was studied. We respectively knocked out there genes from Synechocystis sp. PCC6803and obtained the mutants. We studied the function of mutant△sll0871.The core-membrane linker(LcM) is a multi-domain, multifunctional protein in the PBS. That may act in supporting the core complex formation, anchoring the PBSs to thylakoid membrane surface and transfer the light energy from the PBSs to the reaction centers. Despite the significant role of the Lcm, its research is hampered because the isolated Lcm is extremely unstable and insoluble as like the other linker polypeptides. Here we established a method of three consecutive column chromatography for extracting PCB:ApcE(1-240/△77-153) and PEB:ApcE(1-240/A77-153) with high efficiency. The purity of the proteins was valuated based on the absorbance ratio, A660/A280(PCB) and A580/A280(PEB). Samples were reconstituted in E. coli, firstly purified by Ni2+affinity chromatography after pretreatment. The proteins were further purified by Anion exchange chromatography, and large amount of PCB:ApcE(1-240/△77-153) and PEB:ApcE(1-240/A77-153) could be separated. The purity ratio of the purified PCB:ApcE(1-240/A77-153) and PEB:ApcE(1-240/△77-153) reached1.09(A66o/A280) and1.39(A580/A280), respectively. The isolated proteins could be further purified with single step of gel filtration column chromatography to obtain PCB:ApcE(1-240/△77-153) and PEB:ApcE(1-240/△77-153) with higher purity. The fluorescence quantum yield (φf=0.07PCB andφi=0.63PEB), the extinction coefficient (εPCB=90000M-1cm-1and εPEB=95000M-1cm-1) suggests that the products are in a near native chromophore conformation of Lcm. The purity ratio respectively reached2.54(A660/A280) and2.85(A580/A280)-This extracting method could not only separate large amount of Lcm in short time, but also obtain much higher purity of Lcm, compared with purifying by Ni2+affinity chromatography only.In order to determine the function of the unknow gene ssr2142, sll0524and sll0584, we respectively knocked out the gene ssr2142,sll0524and sll0584. We obtained the mutants and laid a solid foundation for further study of the structure and function of these genes. We focused on the physiology, metabolism, photosynthesis and genetics of△sll0871, in which the gene sll0871was knocked out. In the following aspects of the growth of the different culture conditions, the light use efficiency, the durability against high light, maximal rate of electron transfer, the detection of the state transition and the content of PQ and MSBQ, we found no significant changes. So we draw a conclusion that the deletion of gene sll0871has little effect on the phenotypic character of Synechocystis sp. PCC6803.