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, 327 (5969), 1132-5

Generating a Prion With Bacterially Expressed Recombinant Prion Protein

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Generating a Prion With Bacterially Expressed Recombinant Prion Protein

Fei Wang et al. Science.

Abstract

The prion hypothesis posits that a misfolded form of prion protein (PrP) is responsible for the infectivity of prion disease. Using recombinant murine PrP purified from Escherichia coli, we created a recombinant prion with the attributes of the pathogenic PrP isoform: aggregated, protease-resistant, and self-perpetuating. After intracerebral injection of the recombinant prion, wild-type mice developed neurological signs in approximately 130 days and reached the terminal stage of disease in approximately 150 days. Characterization of diseased mice revealed classic neuropathology of prion disease, the presence of protease-resistant PrP, and the capability of serially transmitting the disease; these findings confirmed that the mice succumbed to prion disease. Thus, as postulated by the prion hypothesis, the infectivity in mammalian prion disease results from an altered conformation of PrP.

Figures

Fig. 1
Fig. 1
In vitro generated rPrP-res. (A) One round PMCA consisted of 48 cycles of sonication (0.5 minute) and incubation (29.5 minute). At the end, 1/10 reaction mixture was transferred to fresh substrate mixture to start a new round (20). PMCA products were digested with 25 μg/ml PK. C, undigested recPrP. (B) Serial PK digestion of PMCA products. (C) PMCA product was separated into supernatant(s) and pellet(p) by a 1-hour 100,000g centrifugation at 4°C. T, total input; +PK, 25 μg/ml PK digested. (D) With normal mouse brain homogenate (NMBH) as substrate, PMCA was performed with or without rPrP-res seed. Product was digested with 100 μg/ml PK. C, undigested NMBH. (E) After rPrP-res infection, SN56 cells were lysed, digested with 25 μg/ml PK, and centrifuged. The PK-resistant PrP in the pellet was detected by immunoblot analysis. Numbers indicates cell passages. C1, undigested SN56 cell lysate. C2, pellet of PK-digested, uninfected SN56 cell lysates. In all panels, PrP was detected by immunoblot analysis with POM1 anti-PrP antibody except for the right panel in (C). PK digestion was carried out at 37°C for 30 minutes (A, B, and C) or 1 hour (D and E).
Fig. 2
Fig. 2
Intracerebral rPrP-res inoculation. (A) After injection, the remaining inocula with or without PK digestion were electrophoresed and PrP was detected by immunoblot analysis with POM1 anti-PrP antibody. The insert is a lighter exposure of undigested inocula. (B) Mice injected with rPrP-res showed indicated signs.
Fig. 3
Fig. 3
Characterization of rPrP-res caused prion disease. Histological analyses of age-and sex-matched control mice (A, C, and E) and rPrP-res injected mice (B, D, and F). Brain sections were stained by hematoxylin and eosin (A) and (B), an anti-GFAP (glial fibrillary acidic protein) antibody (C) and (D), and SAF84 anti-PrP antibody (E) and (F). Immunohistochemical stains were counterstained with hematoxylin. Scale bar represents 50 μm. (G) Brain homogenates of an rPrP-res inoculated mouse or mice inoculated with control inocula 1–3 were digested at 37°C for 1 hour with indicated concentrations of PK. PrP was detected by immunoblot analysis with M20 anti-PrP antibody. (H) Brain homogenates of mice that received second round transmission or control mice inoculated with inoculum diluent (PBS) were digested by 50 μg/ml PK at 37°C for 1 hour. PrP was detected by immunoblot analysis with POM1 antibody. Number indicates the mouse from which the inocula were prepared.

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