A systematic investigation of production of synthetic prions from recombinant prion protein

Abstract

According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre 'synthetic' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20,000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved.

Keywords: prion; prion amyloid; prion disease; prion protein; synthetic prions.

Figure 1.

Plate format for matrix core conditions. Master 96-well plates were prepared so that wells contained α- or β-PrP at a final protein concentration of 0.01 or 0.1 mg ml⁻¹ in a fixed set of core solvent conditions of defined pH and concentration of DTT and NaCl. Condition variables and additives (table 1) were introduced to this master plate format, after which plates were incubated for different time periods at different temperatures (as detailed in Methods) prior to infectivity measurement in the ASCA.

Figure 2.

ASCA measurement of RML prion infectivity. (a) Dose–response curve of RML prion infection of tissue culture wells containing 18 000 PK1/2 cells. The number of mouse intracerebral LD₅₀ units of RML prions applied per well is plotted against mean spot number per well ± s.e.m. (n = 6) determined at the fourth cell split. (b) Representative examples of positive and negative ELISPOT wells.

Figure 3.

Example positive wells from the matrix. ELISPOT wells containing spots with the characteristic visual appearance seen in prion bioassay by SCA were observed to increase successively over cell splits three to five. The matrix conditions that generated these positive findings are detailed next to the wells and were observed in the presence of copper ions at a 5 : 1 molar ratio with PrP and incubation at room temperature (RT) for 1 h (immediate) prior to inoculating the cells. These findings were not consistently reproducible.