Insect Cell-Derived Cofactors Become Fully Functional after Proteinase K and Heat Treatment for High-Fidelity Amplification of Glycosylphosphatidylinositol-Anchored Recombinant Scrapie and BSE Prion Proteins

摘要: The central event in prion infection is the conformational conversion of host-encoded cellular protein (PrPC) into pathogenic isoform (PrPSc). Diverse mammalian species possess cofactors required for vitro replication PrPSc by protein-misfolding cyclic amplification (PMCA), but lower organisms, such as bacteria, yeasts, and insects, reportedly lack essential cofactors. Various components, RNA, lipids, other identified cofactor molecules, are commonly distributed both eukaryotes prokaryotes, reasons absence activity organisms remain to be elucidated. Previously, we reported that brain-derived factors were necessary glycosylphosphatidylinositol-anchored baculovirus-derived recombinant PrP (Bac-PrP). Here, demonstrate following protease digestion heat treatment, insect cell lysates had functional Bac-PrP PMCA. Mammalian seeds Bac-PrPSc generated PMCA using cell-derived showed similar pathogenicity produced very lesions brains inoculated mice. These results suggested high-fidelity present cells was masked or inhibited native state. We suggest not only also DNA, key components PMCA, although expression nucleic acids. substances (iPMCA) highly useful ultrasensitive detection some strains.