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Prion Degradation by a Feather-Degrading Keratinase

Prion Degradation by a Feather-Degrading Keratinase. Jason Shih North Carolina State University. Keratinase Technology. Purified enzyme, MW = 33 kDa Gene isolated and sequenced Genetic modification for over-expression Scale-up production by fermentation Feather process Feed enzyme

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Prion Degradation by a Feather-Degrading Keratinase

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  1. Prion Degradation by a Feather-Degrading Keratinase Jason Shih North Carolina State University

  2. Keratinase Technology • Purified enzyme, MW = 33 kDa • Gene isolated and sequenced • Genetic modification for over-expression • Scale-up production by fermentation • Feather process • Feed enzyme • Prion degradation

  3. Transmissible Spongiform Encephalopathy (TSE): A Timely and Global Issue • Over 180,000 cases of Mad Cow Disease (or Bovine Spongiform Encephalopathy, BSE) have been reported in Europe. • Correlated with cases of Creutzfeldt-Jakob Disease (CJD), a fatal neurodegenerative disease in humans. • No effective diagnostics, vaccines or therapeutics are available for BSE or CJD. • Concerns about BSE and CJD transmission have forced a re-examination of animal feed production and hospital safety measures, respectively.

  4. Prion Structure PrPC PrPSc

  5. keratinase Degradation Treating Prion Proteins with keratinase Conformational Change PrPSc Infectious, insoluble PrPC Non-infectious, soluble

  6. “Proof of Concept” BSE Experiment 1 2 3 4 5 6 7 8 9 10

  7. Brain Stem Tissue Homogenization Heat pre-treatment (115o C - 40 min) Keratinase digestion (50o C - 1hr) MeOH precipitation Redissolved in buffer SDS-PAGE Western blotting and detection

  8. Pre-heat and Keratinase on tissuewhole homogenates and supernatants

  9. Effects ofN-lauroylsarcosine (sarc) and KE

  10. Detergent Effect

  11. Enzymatic degradation of prionsfrom different individual animals

  12. Enzyme Concentrations

  13. Digestion Time

  14. Temperature Effect

  15. Digestion by various enzymes

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