Prions: Methods for Assay, Purification, and Characterization

Prusiner, Stanley B.; McKinley, Michael P.; Bolton, David; Bowman, Karl M.; Groth, Darlene; Cochran, S. Patricia; Hennessey, Elizabeth M.; Braunfeld, Michael B.; Baringer, J. Richard; Chatigny, M. A.

doi:10.1016/b978-0-12-470208-0.50014-1

Cited by 25 publications

(17 citation statements)

References 56 publications

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“…In this present study it was found that the test samples could be injected without dilution if they were at neutral pH. Under these conditions the four logs of scrapie infectivity detected after NaOH exposure (Table 8) approximate the titre of CJD agent which survives a similar treatment [30], and confirm other reports that NaOH treatment is not completely effective with TDE agents [13,16,27,31,33]. An unexpected and unexplained finding in the NaOH experiments was that with 263K(II), BSE(II) and (possibly) ME7, two hour exposures were less effective than those for 30 or 60 min; this was not associated with any significant differences in the pH of samples within the same experiment.…”

Section: Resultssupporting

confidence: 87%

Decontamination studies with the agents of bovine spongiform encephalopathy and scrapie

Taylor

Fraser

McConnell

et al. 1994

Archives of Virology

178

127

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Macerates of bovine brain infected with bovine spongiform encephalopathy (BSE) agent, and rodent brain infected with the 263K or ME7 strains of scrapie agent, were subjected to porous-load autoclaving at temperatures between 134 and 138 degrees C for < or = 60 min. Bioassay in rodents showed that none of the regimens produced complete inactivation. Homogenates of BSE-infected bovine brain were exposed for < or = 120 min to solutions of sodium hypochlorite or sodium dichloroisocyanurate containing < or = 16,500 ppm available chlorine. There was no detectable survival of infectivity after the hypochlorite treatments but none of the dichloroisocyanurate solutions produced complete inactivation. Homogenates of BSE-infected bovine brain, and rodent brain infected with the 263K and ME7 strains of scrapie agent, were exposed for < or = 120 min to 1M or 2M sodium hydroxide but no procedure produced complete inactivation of all agents tested.

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Section: Resultssupporting

confidence: 87%

Decontamination studies with the agents of bovine spongiform encephalopathy and scrapie

Taylor

Fraser

McConnell

et al. 1994

Archives of Virology

178

127

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“…PMCA and serial dilution/propagation experiments were performed as described (22) by using purified substrates instead of brain homogenates. Standard bioassay and neuropathology techniques were used to measure prion infectivity and strain properties (56). PrP Sc stability was assayed as described (46).…”

Section: Methodsmentioning

confidence: 99%

Formation of native prions from minimal components in vitro

Deleault¹,

Harris²,

Rees³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

585

635

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The conformational change of a host protein, PrP C , into a disease-associated isoform, PrP Sc , appears to play a critical role in the pathogenesis of prion diseases such as Creutzfeldt–Jakob disease and scrapie. However, the fundamental mechanism by which infectious prions are produced in neurons remains unknown. To investigate the mechanism of prion formation biochemically, we conducted a series of experiments using the protein misfolding cyclic amplification (PMCA) technique with a preparation containing only native PrP C and copurified lipid molecules. These experiments showed that successful PMCA propagation of PrP Sc molecules in a purified system requires accessory polyanion molecules. In addition, we found that PrP Sc molecules could be formed de novo from these defined components in the absence of preexisting prions. Inoculation of samples containing either prion-seeded or spontaneously generated PrP Sc molecules into hamsters caused scrapie, which was transmissible on second passage. These results show that prions able to infect wild-type hamsters can be formed from a minimal set of components including native PrP C molecules, copurified lipid molecules, and a synthetic polyanion.

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“…In a detailed summary of several different preparative approaches, one group reported 100-1,000-fold specific purifications of protein were typically achieved in complex preparations made with both proteinase K and Mn [27]. The CJD density gradient fractions shown here are comparatively >~ 10,000-fold purified.…”

Section: Discussionmentioning

confidence: 96%

Nuclease treatment results in high specific purification of Creutzfeldt-Jakob disease infectivity with a density characteristic of nucleic acid-protein complexes

Sklaviadis

Akowitz

Manuelidis

et al. 1990

Archives of Virology

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Representative preparations of partially purified Creutzfeldt-Jakob disease (CJD), including disaggregated density gradient fractions, were treated with a variety of nucleases. RNases as well as exhaustive digestions with micrococcal nuclease did not significantly diminish infectivity, but resulted in an approximately 7,000-fold specific purification of infectivity with respect to nucleic acid. Protected nucleic acids included species of up to 2,000 bases in length. After nuclease treatment, infectivity co-migrated with nucleic acid-protein complexes at a density of 1.27 g/cm3 in sucrose. Substantial specific protein purification were also achieved in the gradient step (approximately 11,000-fold), where 70% the host Gp34 ("prion protein") as well as other free proteins separated from infectivity. These CJD purifications are better than those previously attained in scrapie, and may be useful for further studies of non-host protein and nucleic acid species. The data are consistent with the hypothesis that CJD-like agents are composed of nucleic acid-protein complexes.

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Prions: Methods for Assay, Purification, and Characterization

Cited by 25 publications

References 56 publications

Decontamination studies with the agents of bovine spongiform encephalopathy and scrapie

Decontamination studies with the agents of bovine spongiform encephalopathy and scrapie

Formation of native prions from minimal components in vitro

Nuclease treatment results in high specific purification of Creutzfeldt-Jakob disease infectivity with a density characteristic of nucleic acid-protein complexes

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