Prion diseases are the best characterized members of a number of diseases causing neurologic degeneration of the brain. Infectious particles causing scrapie or other prion diseases are composed largely, if not entirely, of an abnormal isoform of a cellular protein denoted PrPSc. In order to analyze how prions enter and spread in the brain, it is important to learn about the cellular proteins interacting with PrP in the normal as well as in the diseased brain. The identification of two PrP ligands of 45 and 110 kDa (Pli 45, Pli 110) has enabled us to investigate these questions. As a first step, it will be required to analyze Plis in molecular detail. This has been accomplished partially for pli 45. Pli 110 will be purified by a combination of detergent extractions, ion exchange chromatography and reverse phase HPLC. Preparative two- dimensional gel electrophoresis, followed by transfer to PVDF membranes, will allow amino acid sequencing. If the N-terminus should be blocked, peptides will be produced by chemical or enzymatic cleavage of Pli 110. Peptides will be separated on reverse phase HPLC and sequenced. In a parallel approach, Pli 110 transferred to nitrocellulose will be injected into rabbits to produce antisera. Specific antibodies will be selected on immobilized Pli 110. If there will be amino acid sequence available, oligonucleotide probes will be synthesized and, after labeling, be used as probes to search a cDNA library made from hamster brain mRNA. Construction of a cDNA library will be according to standard protocols with some improvements. As a vector lambdazap will be used. In addition to the advantage of high efficiency and rapid subcloning, the cDNA can also be expressed as a fusion protein with beta-galactosidase. This will allow us to search for specific clones by immunoscreening with the rabbit antisera mentioned above. Direct binding of radiolabelled PrP to fusion proteins will be another strategy to identify specific clones encoding Plis. Final proof for the cloned cDNAs and Plis will be sought either by hybrid selection of mRNA encoding Plis, or by direct expression of full length cDNAs in eukaryotic cells.
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