Abstract
Protein misfolding cyclic amplification (PMCA) recapitulates the prion protein (PrP) conversion process under cell-free conditions. PMCA was initially established with brain material and then with further simplified constituents such as partially purified and recombinant PrP. However, availability of brain material from some species or brain material from animals with certain mutations or polymorphisms within the PrP gene is often limited. Moreover, preparation of native PrP from mammalian cells and tissues, as well as recombinant PrP from bacterial cells, involves time-consuming purification steps. To establish a convenient and versatile PMCA procedure unrestricted to the availability of substrate sources, we attempted to conduct PMCA with the lysate of cells that express cellular PrP (PrPC). PrPSc was efficiently amplified with lysate of rabbit kidney epithelial RK13 cells stably transfected with the mouse or Syrian hamster PrP gene. Furthermore, PMCA was also successful with lysate of other established cell lines of neuronal or non-neuronal origins. Together with the data showing that the abundance of PrPC in cell lysate was a critical factor to drive efficient PrPSc amplification, our results demonstrate that cell lysate in which PrPC is present abundantly serves as an excellent substrate source for PMCA.
Highlights
Conformational conversion of the a helix rich cellular prion protein (PrPC) to the b sheet rich scrapie prion protein (PrPSc) is the major biochemical event that characterizes prion diseases [1]
The current study demonstrates that cell lysate with concentrated PrPC allowed robust Protein misfolding cyclic amplification (PMCA) of PrPSc from multiple strains and species
The ability of cell lysate to support PrPSc formation in PMCA is comparable to that of wild type brain material. This result suggests that cell lysate can replace animal organ-derived material for in vitro PrPSc amplification without compromising PrP conversion efficiency
Summary
Conformational conversion of the a helix rich cellular prion protein (PrPC) to the b sheet rich scrapie prion protein (PrPSc) is the major biochemical event that characterizes prion diseases [1]. PrP conversion in cultured cells and animal models is possible, it has been quite difficult to reproduce the process in vitro. Protein misfolding cyclic amplification (PMCA) is an assay that mimics the PrPSc propagation process under cell-free conditions. This method amplifies misfolded PrP by converting PrPC to PrPSc during incubation with periodic sonication [4]. PrPSc generated by PMCA is infectious in wild-type animals [5] and can be indefinitely propagated with preserved properties of the original PrPSc [5,6,7]. PMCA is quite useful in studying the cofactors that influence PrP conversion [14,15,16,17,18,19,20,21,22,23,24], and in detecting PrPSc from biological samples of humans and animals [25,26,27,28,29,30,31,32,33,34,35,36,37]
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