Enhancement of protein misfolding cyclic amplification by using concentrated cellular prion protein source

Abstract

Protein misfolding cyclic amplification (PMCA) is a cell-free assay mimicking the prion replication process. However, constraints affecting PMCA have not been well-defined. Although cellular prion protein (PrP(C)) is required for prion replication, the influence of PrP(C) abundance on PMCA has not been assessed. Here, we show that PMCA was enhanced by using mouse brain material in which PrP(C) was overexpressed. Tg(MoPrP)4112 mice overexpressing PrP(C) supported more sensitive and efficient PMCA than wild type mice. As brain homogenate of Tg(MoPrP)4112 mice was diluted with PrP(C)-deficient brain material, PMCA became less robust. Our studies suggest that abundance of PrP(C) is a determinant that directs enhancement of PMCA. PMCA established here will contribute to optimizing conditions to enhance PrP(Sc) amplification by using concentrated PrP(C) source and expands the use of this methodology. (C) 2009 Elsevier Inc. All rights reserved.