Screening Interacting Proteins With Deinococcus Radiodurans Peculiar Protein PprI

Background and purpose:All species in the genus Deinococcus, in particular Deinococcus radiodurans (DR), are extremely resistant to a number of agents and conditions that damage DNA, including ionizing and ultraviolet (UV) radiation and hydrogen peroxide. The research of pprI Mutant show that pprI plays key role in D.radiodurans'extremely resistance to radiation. Thus ,this study will screening interacting proteins with Deinococcus radiodurans peculiar Protein PprI , to understand the complicated PprI regulation network of the DNA damage restoration system in D. radiodurans, will make for identifying the mechanisms of D. radiodurans'extremely resistance to radiation.Methods: PGBKT7-pprI and PGADT7-pprI were constructed for yeast two–hybrid,the combinant plamids pGBKT7–pprI and pGADT7 were co–transefored into yeast srtain AH109 and its expression products were assayed by using western blot 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 ,Screening to isolate proteins that might interact with PprI. Later, the interaction of one screend proteins were checked with co–immunopricipitation experiment. To analyze domains of 10 screened proteins and predicte interaction chain of PprI and SCS by Using CDD and SMART online software.Results:The yeast two hybrid bait vector pGBKT7–pprI have been constructed, Western blotting confirmed the expression of fusion protein in the AH109 and the pGBKT7–pprI was not toxic to AH109 ;screened ten positive blue clones by using yeast two-hybrid; Co-immunoprecipitation showed that when HA-SCS was immunoprecipitated by anti-HA polyclonal antibody,Myc-pprI was identified by western blotting with anti-Myc monoclonal antibody from immunoprecipitated complex,and vice versa; Bioinformatics analysis show that 4 screened proteins contain the same NADB domain.Conclusions:(1)The yeast two hybrid bait vector pGBKT7–pprI have been constructed,the self-activating experiment show that pGBKT7–pprI can not activate by itself;(2)Ten proteins were screended from D.radiodurans genomic expression library ;(3)Co-immunopricipitation detection show that PprI and SCS can combine together, PprI may affect the energy Survival system of D. radiodurans.(4)Bioinformatics analysis show that PprI may regulate the expression amount and activation of oxidoreductase through interacting with NADB domain.