Ablation of prion protein immunoreactivity by heating in saturated calcium hydroxide

<p>Abstract</p> <p>Background</p> <p>Prions, the infectious agents that cause transmissible spongiform encephalopathies (TSEs), are relatively resistant to destruction by physical, enzymatic, and chemical treatments. Hydrolysis in boiling saturated calcium hydroxide (limewater) utilizes inexpensive chemicals to digest protein components of offal. The purpose of this work was to determine if incubating brain material from scrapie-infected sheep in near-boiling saturated calcium hydroxide solution (Ca(OH)₂) would abolish immunoreactivity of the infectious prion (PrP^Sc) as determined by western blot.</p> <p>Findings</p> <p>After incubating for as few as 10 minutes in saturated calcium hydroxide at 99°C, immunoreactivity of protease resistant bands by western blot analysis is completely lost.</p> <p>Conclusion</p> <p>Boiling in limewater may offer an alternative for disposal of carcasses and enable alternative uses for rendered products from potentially infected carcasses.</p

Diffusion of Protease into Meat & Bone Meal for Solubility Improvement and Potential Inactivation of the BSE Prion

BACKGROUND: Government-imposed feed bans have created a need for new applications for meat & bone meal (MBM). Many potential new applications require MBM protein to be both soluble and free of infectious prion. Treatment with protease is generally effective in reducing insoluble, thermally-denatured proteins to soluble peptides. It has been reported in the literature that certain proteases, including Versazyme™, are able to degrade infectious prions in a system where the prions are readily accessible to proteolytic attack. Prions distributed within MBM, however, may conceivably be protected from proteases. METHODOLOGY/PRINCIPAL FINDINGS: The overall rate of proteolytic MBM digestion depends greatly on whether the protease can penetrate deep within individual particles, or if the protease can only act near the surface of the particle. This research examined the barriers to the diffusion of Versazyme™ into particles of MBM. Confocal microscopy demonstrated differences in the density distributions between the bone and the soft tissue particles of MBM. By tracking the diffusion of fluorescently labeled Versazyme™ through individual particles, it was found that bone particles show full Versazyme™ penetration within 30 minutes, while penetration of soft tissue particles can take up to four hours, depending on the particle's diameter. From the variety of normal proteins comprising MBM, a specific protein was chosen to serve as a prion surrogate based on characteristics including size, solubility, distribution and abundance. This surrogate was used to measure the effect of several factors on Versazyme™ diffusion. CONCLUSIONS/SIGNIFICANCE: Results showed that surrogate distributed in bone particles was more susceptible to degradation than that in soft tissue particles. Three factors controllable by unit operations in an industrial-scale process were also tested. It was found that removing the lipid content and hydrating MBM prior to incubation both significantly increased the rate of surrogate degradation. In a test of particle size, the smallest collected diameter range demonstrated the largest degradation of the prion surrogate, suggesting milling would be beneficial

Enzymatic Degradation of PrPSc by a Protease Secreted from Aeropyrum pernix K1

BACKGROUND: An R30 fraction from the growth medium of Aeropyrum pernix was analyzed for the protease that can digest the pathological prion protein isoform (PrP(Sc)) from different species (human, bovine, deer and mouse). METHODOLOGY/PRINCIPAL FINDINGS: Degradation of the PrP(Sc) isoform by the R30 fraction and the purified protease was evaluated using the 6H4 anti-PrP monoclonal antibody. Fragments from the N-terminal and C-terminal of PrP(Sc) were also monitored by Western blotting using the EB8 anti-PrP monoclonal antibody, and by dot blotting using the C7/5 anti-PrP monoclonal antibody, respectively. For detection of smaller peptides from incomplete digestion of PrP(Sc), the EB8 monoclonal antibody was used after precipitation with sodium phosphotungstate. Characterization of the purified active protease from the R30 fraction was achieved, through purification by fast protein liquid chromatography, and identification by tandem mass spectrometry the serine metalloprotease pernisine. SDS-PAGE and zymography show the purified pernisine plus its proregion with a molecular weight of ca. 45 kDa, and the mature purified pernisine as ca. 23 kDa. The purified pernisine was active between 58 °C and 99 °C, and between pH 3.5 and 8.0. The temperature and pH optima of the enzymatic activity of the purified pernisine in the presence of 1 mM CaCl(2) were 105 °C ± 0.5 °C and pH 6.5 ± 0.2, respectively. CONCLUSIONS/SIGNIFICANCE: Our study has identified and characterized pernisine as a thermostable serine metalloprotease that is secreted from A. pernix and that can digest the pathological prion protein PrP(Sc)

Enzymatic formulation capable of degrading scrapie prion under mild digestion conditions

The prion agent is notoriously resistant to common proteases and conventional sterilisation procedures. The current methods known to destroy prion infectivity such as incineration, alkaline and thermal hydrolysis are harsh, destructive, environmentally polluting and potentially hazardous, thus limit their applications for decontamination of delicate medical and laboratory devices, remediation of prion contaminated environment and for processing animal by-products including specified risk materials and carcases. Therefore, an environmentally friendly, non-destructive enzymatic degradation approach is highly desirable. A feather-degrading Bacillus licheniformis N22 keratinase has been isolated which degraded scrapie prion to undetectable level of PrPSc signals as determined by Western Blot analysis. Prion infectivity was verified by ex vivo cell-based assay. An enzymatic formulation combining N22 keratinase and biosurfactant derived from Pseudomonas aeruginosa degraded PrPSc at 65°C in 10 min to undetectable level -. A time-course degradation analysis carried out at 50°C over 2 h revealed the progressive attenuation of PrPSc intensity. Test of residual infectivity by standard cell culture assay confirmed that the enzymatic formulation reduced PrPSc infectivity to undetectable levels as compared to cells challenged with untreated standard scrapie sheep prion (SSBP/1) (p-value = 0.008 at 95% confidence interval). This novel enzymatic formulation has significant potential application for prion decontamination in various environmentally friendly systems under mild treatment conditions

Hydrolysis of the amyloid prion protein and non pathogenic meat and bone meal by anaerobic thermophilic prokaryotes and Streptomyces subspecies

International audienc

Utilisation des microorganismes dans l'hydrolyse des amyloïdes et des farines animales

National audienceA la suite de l'interdiction des farines animales en alimentation animale, il est nécessaire de mettre au point des procédés simples et économiques pour les valoriser et les décontaminer vis-à-vis de la présence possible de prion pathogène. L'usage des microorganismes est une solution possible. Aussi, des collections de microorganismes capables de croître sur la kératine ou sur des farines animales et sécrétant des protéases capables de dégrader la PrPsc contenue dans les farines animales ont été criblées. Ceci a permis de découvrir trois souches de bactéries thermophiles, isolées de différentes sources chaudes réparties sur la planète, qui sont capables de dégrader la protéine prion infectieuse PrPsc et de croître sur un milieu composé de farines animales. Leur activité protéolytique, de type chymotrypsique pour l'essentiel, est maximale à la température de 60 à 80°C et focalisée sur certaines liaisons peptidiques qui sont nombreuses sur la protéine prion. Leur action découpe ainsi la protéine prion en morceaux plus courts inoffensifs. Les perspectives sont de mettre en oeuvre ces microorganismes thermophiles dont le patrimoine protéolytique permettrait la dégradation des farines animales, actuellement incinérées, et les protéines prions qu’elles renferment