Screening For Proteins Interacted With PrP In Mouse Brain CDNA Library

Transmissible spongiform encephalopathy (TSE) is a human and animal's central nervous system degenerative disease caused by the Scrapie Prion Proein (PrPSC) which is the isoform of the Celluar Prion Protein (PrPC). Several TSEs have been reported, such as Creutzfeldt-Jakob disease (CJD), Kuru disease (Kuru), Transmissible mink encephalopathy (TME), Cervine chronicity wasting disease (CWD), Bovine spongiform encephalopathy (BSE).The typical clinical symptoms of TSE include progressive ataxia, thrill, unsteadiness, dementia, perceptual hyperesthesia, abnormal behavior. 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. Some researchers presume that there may be some other proteins affect in the pathogenesy of TSE, but these proteins have not been defined.PrPC is a high conservative protein that anchors to the cell membrane surface by glycosyl-phosphatidyl inositol anchor (GPI anchor) most expressed in neuron. The physiology function of PrPC is still unclear. But some studies have proved that PrPC is a copper-binding protein, so it may have the activity of superoxide dismutase (SOD). In order to find the function of PrPC, several associated proteins with PrPC have been studied, for instance, mouse STLI which is related to mouse PrPC, synapsin I, Grb-2, Pint 1, N-CAMs, immunoglobulin C2, and 37-kDa/67-kDa LRP which is the acceptor of the human PrPC. Most of these proteins are reside in the cellular membrane, endocytosis interval or secretory pathway, which means that PrPC may act an important role in the signal transduction, neuro-syncretio and axonal process of growth. 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 large scale and high-flux technologies can be used to study protein-protein interaction, such as protein chip, biology mass spectra, bioinformatics analysis, yeast two-hybrid system. Yeast two-hybrid system has became one of the most drastic methods in the study of protein-protein interaction.Traditional yeast two-hybrid system is established on the nature of GAL4. At the last 20 years, this system became better improved and developed, but the proteins analyzed in this system is localized in nucleolus only, which cause a impossible overcome shortage of this system. For instance, many proteins do not have biological activity after the post-translational processing (such as, glycosylation, formation of disulidelinkage) in endoplasmic reticulum or some other organellas, but these proteins may be impossible to get into the nucleolus, or even these proteins can not be post-translational processed, so it is unsuitable to study these proteins in the traditional yeast two-hybrid system. Some interactions between protein and protein may only reside in cytoplasm, they may fail to interact to each other in the nucleolus and some proteins are activated by transcription factors (ATF) or suppressor factors which can regulate report gene, so it is obviously unsuitable to study these proteins in the traditionary yeast two-hybrid system. In addition, expression of the foreign proteins may exhibit toxicity to the yeast cell, and functional incapacitation of the proteins for its fault-folding, also limited the application of the traditional yeast two-hybrid system.Stratagene CytoTrapTM two-hybrid system is based upon generating fusion proteins whose interaction in the yeast cytoplasm activates the Ras-signaling pathway, inducing cell growth, so it is also called as SOS restorey system. Huaman Sos (hSos) protein anchored in the inboard cellular membrane can activate the Ras-signaling pathway, and cdc25 is the yeast homologue of the huaman Sos (hSos) gene. The CytoTrapTM two-hybrid system uses the yeast temperature-sensitive mutant strain cdc25H which contains a point mutation at amino acid residue 1328 of the cdc25 gene. The cdc25 mutation in the cdc25H strain prevents growth at 37℃, but allows normal growth at the permissive temperature (22℃～25℃).In the CytoTrapTM two-hybrid system, expression of hSos and its subsequent localization to the plasma membrane allows the cdc25H yeast strain to grow at 37℃. 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 was amplified by the method of PCR and subcloned into the pSos plasmid. The recombinant bait vector pSos-PrP23-231 of yeast two -hybrid system was obtained after verified by endonucleases digestion analysis. Meanwhile, control co-transformation experiments were done using the pSos-PrP23-231 and the pMYR empty vector to test the self-activation ability of the bait vector. The expression of the fusion protein and toxic effect to the yeast cell were detected through transformation of the bait plasmid and Western blot analysis. The results showed that the bait vector with the PrP23-231 domain of mouse in yeast two-hybrid system was constructed successfully. The PrP23-231 was not toxic to cdc25H and does not have self-activation. The CytoTrap yeast two-hybrid system could be used to fish the interacting proteins with PrP23-231 domain of mouse prion, which supplied further study of the function and fault-folding mechanism of prion.Commercialize mouse brain cDNA library was subpackaged and cDNA library plasmid were amplified and extracted. The concentration and purity of the cDNA library plasmid were also measured (66μg/mL, OD260/OD280=1.77), which was suitable for the transformation experiment. In the screening experiment, the pSos-PrP23-231 and cDNA library plasmid were co-transformed into the cdc25Hαyeast competent cell by the co-transforming method, and experiment was done to test the temperature-sensitive revert of the yeast competent cell. The result indicated that the mutation stains can be used in screening experiment .At last, 1 positive clone was picked up among 26 putative positive clones after verification. By the extracting the plasmid from the positive clones, endonucleases digestion analysis and sequencing, it showed that it had high sequence homology to the sequence of PrP. The result of the screening experiment further confirmed that prion protein may exist as the dimer or multimer in physiological state...