Screening And Identification Of Proteins That Interact With Peroxiredoxin1from Dunaliella Salina

BackgroundPeroxiredoxins （Prdxs） are antioxidant proteins that are distributed ubiquitously inhigh level, they can protect cells from damage by catalyzing peroxide reduction ofH2O2, peroxynitrite and hydroperoxides. The molecular weight of Prdxs is between22kDa and27kD. Prdxs possess six mammalian isoforms which can classified intothree subgroups based on the number and position of conserved Cys residues:1.Typical2-Cys Prdxs;2. Atypical2-Cys Prdxs;3.1-Cys Prdxs. Peroxiredoxin1(Prdx1) is the most extensive distributed member of the Prdxs family and belongs tothe typical2-Cys Prdxs. Prdx1is known to be involved in both antioxidant and avariety of biological processes including cell growth, cell proliferation, celldifferentiation and cell apoptosis. Moreover, there are also some evidences showedthat the expression of Prdx1is aberrant increased in many kinds of tumors and Prdx1modulates cell signaling of the development of tumors. Therefore, as the molecularmediator, the function of Prdx1has received attention.ObjectiveTo explore the molecular mechanism of Prdx1in Dunaliella salina, this studyscreened the proteins that could interact with Prdx1in Dunaliella salina and verifiedthis interaction through cotransformation experiment and in-vitro experiments.Methods1. Screening proteins that can interact with Prdx1in Dunaliella salina by yeast two-hybrid system.The open reading frame (ORF) of Prdx1gene was obtained by PCR and then wasinserted into pGBKT7vector after being identified by DNA sequencing. Therecombinant plasmid pGBKT7-Prdx1was transformed into yeast strains Y187andAH109respectively by using PEG/LiAc method. Then the transcriptional activationand toxicity of the bait plasmid pGBKT7-Prdx1were tested to identify if the baitprotein was suitable to be used in the yeast two-hybrid system.The vector pGBKT7-Prdx1was used as the bait to screen the proteins that caninteract with Prdx1from the cDNA library of Dunaliella salina. The positivecolonies were obtained after being screened by auxotroph culture andα-galactosidase activity.2. Identification of the interactionCotransformation experiment: The prey plasmid of the positive colony was extractedand then was cotransformed into the yeast strain AH109with the bait plasmid. Theinteraction was identified by auxotroph culture and α-galactosidase activity.In-vitro experiments: The total RNA of Dunaliella salina were extracted andincubated with Prdx1to test the interaction by agarose gel shift assay and protectionof Prdx1-nucleic acid complexes.Results1. Screening proteins that can interact with Prdx1in Dunaliella salina by Yeasttwo-hybrid system.The ORF of Prdx1was obtained and the bait plasmid pGBKT7-Prdx1wasconstructed successfully. The fusion plasmid pGBKT7-Prdx1was transformed intoyeast strains Y187and AH109respectively. After being tested by the transcriptionalactivation and toxicity assay, the bait protein was both inactive and nontoxic.Therefore, the constructed plasmid can be used in the yeast two-hybrid system.The yeast two-hybrid system was used to screen the interacting proteins from thecDNA library of Dunaliella salina. After screening,168positive colonies wereobtained including Dunaliella salina28s RNA. As a member of Peroxiredoxins, thefunctions of Prdx1involve in both eliminating peroxide and regulating multiplebiological processes as the molecular mediator. However, studies about if Prdx1 involve in the RNA metabolism. Because it is very difficult to isolate28s RNA, thusRNA was selected as the research object.2. Identification of the interactionCotransformation experiment: Extracting prey plasmid pGADT7-RNA andcotransforming the prey plasmid into the yeast strain AH109with the bait plasmidpGBKT7-Prdx1. The cotransformation can survive on the auxotrophic base andexpress the blue phenotype, the results showed that Prdx1can interact with RNA inthe yeast strains.In-vitro experiment: The vector pET28(a)+-Prdx1was constructed and transformedinto E.coli BL21to express Prdx1protein. Then the protein was purified by Ni-IDASefinose Kit. The total RNA of Dunaliella salina were extracted and incubated withpurified Prdx1protein to conduct agarose gel shift experiment and protection ofPrdx1-nucleic acid complexes. The test also identified the interaction between Prdx1and RNA.ConclusionPrdx1of Dunaliella salina was used as the bait protein to screen the cDNA library ofDunaliella salina. After screening, the cotransformation experiment and the in-vitroexperiment were used to identify the interaction. Protection of Prdx1-nucleic acidcomplexes suggested that Prdx1can protect RNA in RNA metabolism.