Screening For Proteins Interacted With PrP Using Yeast Two-Hybrid In Mouse Brain CDNA Library

Transmissible spongiform encephalopathy (TSE) is a human and animal's central nervous system degenerative disease. The typical feature is the acumulation of PrPsc which is abound with theβ-fold and possesses partial protease resistance according to the histopathology. PrPsc is from the PrPc that is abound withα-Helix, which mechanism is still unknown. PrPc is a high conservative protein that anchors to the cell membrane surface by glycosyl-phosphatidyl inositol anchor (GPI anchor) most expressed in neuron.Some researchers presume that there may be some other proteins affect in the pathogenesy of TSE, so searching for the proteins interacted with PrPc is significant for the research of the transform mechanism, and also the physiology function of PrPc.There are several technologies can be used to study protein-protein interaction, Yeast two-hybrid system has became one of the most drastic methods in the study of protein-protein interaction.The CytoTrap system uses the yeast S. cerevisiae temperature-sensitive mutant strain cdc25H,The cdc25 mutation present in the cdc25H strain prevents growth at 37℃, but allows normal growth at the permissive temperature (25℃). The CytoTrap system is based on the ability of the human Sos protein (hSos), to complement the cdc25 defect and to activate the yeast Ras-signaling pathway. Expression of hSos and its subsequent localization to the plasma membrane allows the cdc25H yeast strain to grow at 37℃. The localization of hSos to the plasma membrane occurs through the interaction of two-hybrid proteins. All the interactions of protein-protein happen in cytoplasm, so it can overcome the limitations of the traditionary yeast two-hybrid system.The prion gene subcloned into the pSos plasmid. The expression of the fusion protein PrP23-231 was not toxic to cdc25H and does not have self-activation were detected through transformation of the bait plasmid. The Cyto Trap yeast two-hybrid system could be used to fish the interacting proteins with PrP23-231 domain of mouse prion;At last, 1 positive clone was picked up among 10 putative positive clones after verification. By the extracting the plasmid from the positive clones, endonucleases digestion analysis and sequencing, it showed that Ras protein-specific guanine nucleotide-releasing factor 1(Ras-grf1).In order to validate interaction between protein Ras-grf1 and PrP23-231, we amplified co-immunoprecipitation to further validate interaction. The discoveries of the interaction protein may have important significances for the research of the transform mechanism of PrPc to PrPsc and elucidating the physiology function function of PrP.