Copyright X 1992, American Society for Microbiology Attempts To Convert the Cellular Prion Protein into the Scrapie Isoform in Cell-Free Systems

The scrapie prion protein (PrPFs) is derived from a cellular isoform (PrPC) that acquires protease resistance posttranslationally. We have used several different experimental approaches in attempts to reconstitute in vitro the processes leading to protease-resistant PrPs5 molecules. In the first study, we performed mixing experiments by adding mouse PrP 27-30 (MoPrP 27-30), the protease-resistant core of PrP&, to PrPc and then incubating the mixture to investigate the possibility of heterodimer formation as a first step in prion replication. We used epitopically tagged PrP molecules, synthesized in murine neuroblastoma (N2a) cells transfected with the chimeric mouse/Syrian hamster MHM2 PrP construct, which are recognized by the Syrian hamster-specific monoclonal antibody 3F4. After as long as 24 h of incubation, the reaction mixture was assayed for heterodimeric intermediates of MHM2 PrPc and MoPrPsc and for protease-resistant 3F4-reactive PrP. We were unable to identify any aggregates ofMHM2 PrPc and MoPrP"c on immunoblots; furthermore, we did not observe de novo formation of protease-resistant MHM2 PrP. In a second study, MoPrPc was metabolically radiolabeled in scrapie prion-infected N2a cultured cells, and then the cell extract was homogenized and incubated under various conditions to allow for the formation of protease-resistant MoPrPSc. We observed no radiolabeled MoPrFsc by immunoprecipitation after as long as 24 h of in vitro incubation. In a third approach, Syrian hamster PrP (SHaPrP) was synthesized in a cell-free translation system supplemented with microsoma