Prions Ex Vivo: What Cell Culture Models Tell Us about Infectious Proteins
Figure 3
Generation of infectious protein polymers from minimal components in vitro. (a) For the generation of infectious PrP aggregates, purified recombinant murine PrP comprising amino acid residues 23 to 230 is mixed with lipid POPG and total liver RNA or synthetic polyA RNA. In a so-called protein misfolding cyclic amplification reaction (PMCA), intermittent pulses of sonication shear aggregates and increase the number of seeds. Preformed seeds are subsequently elongated and fragmented by serial dilution of samples into a new reaction buffer (supplemented with recombinant PrP and cofactors) and PMCA. Synthetic prions generated by this method have been shown to chronically infect SN56 and CAD cells [8, 62]. (b) The formation of Sup35 NM fibrils does not require additional cofactors. Purified recombinant NM is diluted to a concentration of 10 μM in phosphate buffered saline and left at room temperature overnight. Formation of NM fibrils is expedited by agitation. Addition of these fibrils to N2a cells stably expressing soluble NM induces a heritable NM prion phenotype [57, 58].
