Screening Interacting Proteins Of Deinococcus Radiodurans Dps-2

Background and purpose: Deinococcus radiodurans (DR) is one of the most powerful biological anti-radiation,known to the world for its ionizing radiation, ultraviolet light, strong oxidizing agents and chemical mutagens of the super-famous for tolerance and resistance . Its resistance mechanism is mainly attributed to its special physical structure and the means to survival, to the precise and efficient DNA damage repair, as well as effective anti-oxidation system. Dps (DNA protection during starvation) is a essential component of anti-oxidation system in Deinococcus radiodurans, there are two kinds of Dps proteins in the DR strain, coded by DR2263 and DRB0092 genes respectively (most bacterias only express one kind of Dps protein ) ,and, the two kinds of Dps protein of DR in the sequence homology comparison of bioinformatics such as the difference between speculation Dps protein in the DR exercise the functions and role of the channels will work with other species are different: the difference between all these is to explain the ultra-strong resistance mechanism in DR will have important significance. The DRB0092(dps) (encoding Dps-2) as an object to study. Constructing the yeast two hybrid bait vector pGBKT7–dps to screen interacting proteins with Deinococcus radiodurans Dps-2 by the yeast two–hybrid. To understand the Dps-2 protein may interact with protein factors which together accomplish the above antioxidant. This will help clear the efficient antioxidant system molecular mechanism for the effective radiation resistance of DR complex regulatory networks and provides new information.Methods: Construct the bait plasmid pGBKT7-dps using by genetic engineering methods, the sequencing is correct, then the combinant plamids pGBKT7–dps and pGADT7 were co–transefored into yeast srtain AH109, to observe whether it can affect the growth of yeast cells and activate the reporter genes. Base on the two genomic libraries (pGADT7–DNADR/Sau3AI and pGADT7–DNADR/HpaII) which were constructed previously ,screened to isolate proteins that might interact with Dps-2 by yeast two-hybrid technology.Results: The plasmid pGBKT7-dps constructed successfully ,the result shows that Dps-2 was not toxic to AH109 and could not activate the reporter gene. The result also demonstrated homotypic interaction of Dps-2 in transformed yeast cells. 7 positive clones were picked out through yeast two-hybrid system.Conclusions: 1.The plasmid pGBKT7-dps constructed successfully in this study, which could not activate the reporter gene. 2. The results demonstrated homotypic interaction of Dps-2 and it can form dimers in vivo. 3. Seven proteins were screended from D.radiodurans genomic expression library, one of these peptides contains the parts of unique genes: DR₂585 and DR₂586, the function of these meaningful peptides concentration related in metabolism of biological macromolecules, combined and degradation.