Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2001-04-12
2003-09-02
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C424S438000, C424S442000, C435S031000, C435S183000
Reexamination Certificate
active
06613505
ABSTRACT:
FIELD OF THE INVENTION
The present invention generally relates to a composition and method for destruction of infectious prion proteins. More specifically, the invention relates to application of proteases for destruction of infectious prion proteins, such as in animal tissue containing prion proteins associated with transmissible spongiform encephalopathy (TSE), e.g., bovine spongiform encephalopathy (BSE) and sheep scrapie. The invention has application to processing of animal meat for human food and animal by-product for animal feed.
BACKGROUND OF THE INVENTION
Prion proteins are conformationally anomalous proteins that are associated with infectious neurodegenerative diseases in human as well as non-human mammalian species.
Prion diseases in non-human mammalian species include scrapie (sheep), transmissible mink encepohalopathy (minks), chronic wasting disease (elk, deer), bovine spongiform encephalopathy (BSE) (cows), feline spongiform encephalopathy (cats), and simian spongiform encephalopathy (monkeys).
In humans a variety of neurodegenerative conditions are etiologically associated with prion proteins, including Creutzfeldt-Jakob disease, Gerstmann-Straüssler-Scheinker syndrome, fatal insomnia, kuru, and variant Creutzfeldt-Jakob disease. Pathogenesis of human prion diseases is associated with carnivorism (BSE-infected beef, causing new variant Creutzfeldt-Jakob disease), administration of human growth hormone (causing iatrogenic Creutzfeldt-Jakob disease) and ritualistic cannibalism (causing kuru).
Over 180,000 BSE cases and 100 human Creutzfeldt-Jakob disease cases have been reported in Europe since 1992, and the human cases are predicted to significantly rise. The spread of such disease is difficult to contain, since such disease has no cure and the pathogenic prion protein is recalcitrant and non-immunogenic. The pathogenic and infectious isoform of prion protein is very stable, rich in &bgr;-sheet structure, and resistant to heat and common proteolytic enzymes (Prusiner, S. B.,
Proc. Natl. Acad. Sci. U.S.A.,
95, 11363 (1998); Cohen, F. E. and Prusiner, S. B.,
Ann. Rev. Biochem.,
67, 793 (1998); and Pan, K-M, Baldwin, M., Nguyen, J., Gasset, M., Serban, A., Groth, D., Mehlhorn, I., Huang, Z., Fletterick, R. J., Cohen, F. E., and Prusiner, S. B.,
Proc. Natl. Acad. Sci. U.S.A,
90, 10962 (1993)).
Significant efforts have been focused on studies of BSE and prion protein contamination of human food supplies deriving from bovine sources, and of prion protein disease generation and propagation in bovine species. Infection in bovine populations has been associated with feeding of bovine herds with feedstocks containing bone meal and rendered organs and tissue deriving from infected cows, sheep and other ruminant animals.
At present, in many countries, animal products that otherwise would provide human food, and animal by-product that otherwise would provide a viable source of raw materials and nutritional supplements for animal feeds, are being incinerated and the ash residue buried, to preclude transmission of prion protein infection deriving from the presence of infectious prion proteins in associated animals.
In Europe, meat bone meal from animal by-products has been banned for feed use. In the United States, no outbreak of BSE has been reported, however, animal and rendering industries have been placed under restrictive regulations to prevent the incidence and spread of disease (Franco, B. A.,
Feed Stuffs, Feb.
12, 2001). Further, the United States has banned imports of meat and meat by-products.
A variety of tests for determining the presence of infectious prion proteins in animal tissue have been developed, including Western blot tests, sandwich immunoassay tests, ELISA tests, fluoroimmunoassay tests, capillary immuno-electrophoresis tests, and plasminogen binding tests (Genetic Engineering News, Vol. 21, No. 6, Mar. 15, 2001), but corresponding capability for industrially-applicable removal of infectious prion proteins from infected animal tissue has not evolved to date.
Infectious prion proteins are resistant to destruction by conventional methods that denature and otherwise degrade conformationally normal proteins, including methods such as autoclaving (even temperatures as high as 200° C. are not effective to inactivate infectious prion proteins), boiling, freezing, and exposure to reagents such as formaldehyde, carbolic acid and chloroform. Typically, incineration or treatment with bleach is employed to destroy the pathogenic isoform of the prion protein.
It therefore would be a significant advance in the art to provide a composition and methodology for destruction of infectious prion proteins, which is applicable to the treatment of biological materials, e.g., animal tissue containing or contaminated with infectious prion proteins.
SUMMARY OF THE INVENTION
The invention provides a method and composition for destruction of infectious prion proteins in tissue containing or contaminated with same.
In one aspect, the invention relates to a method of treatment of tissue associated with, i.e., containing or contaminated with, infectious prion protein (PrP
Sc
). The method comprises the steps of:
(a) heating the tissue to a sufficient temperature and for sufficient time to enhance the proteolytic degradability of infective prion protein associated with the tissue; and
(b) exposing the heated tissue to a proteolytic enzyme that is effective for at least partial reduction of infective prion protein associated with such tissue.
Another aspect of the invention relates to a method of enhancing degradability of an infectious prion protein by proteolytic enzymatic degradation treatment, including (a) heating the prion protein to a temperature below the pyrolytic destruction temperature of the prion protein (i.e., the temperature (>>200° C.) that is normally employed for incineration of the prion protein), followed by (b) enzymatic degradation treatment of the prion protein.
In a further aspect, the invention relates to a method of removing infective prion protein from bovine tissue containing or contaminated with same, the method including cooking the bovine tissue at a temperature in a range of from about 100° C. to about 150° C., e.g., for a time of from about 5 minutes to about 5 hours, followed by (b) exposing the bovine tissue to a proteolytic enzyme at a temperature in a range of from about 35° C. to about 100° C. at which the proteolytic enzyme is thermally stable and proteolytically effective to at least partially destroy the infective prion protein associated with the bovine tissue.
A further aspect of the invention relates to a method of degrading infectious prion protein, comprising (a) heating the infectious prion protein to temperature in a first elevated temperature range, followed by (b) cooling the infectious prion protein to lower elevated temperature in a second elevated temperature range, and (c) exposing the infectious prion protein to a proteolytic enzyme effective at such lower elevated temperature to degrade the infectious prion protein to a benign degradation product.
A still further aspect of the invention relates to a method of at least partially degrading infectious prion protein in tissue containing or contaminated with same, by steps including heating the tissue and simultaneously exposing same to a thermal stable proteolytic enzyme, at sufficient temperature and for sufficient time to at least partially degrade the infectious prion protein.
In another particular aspect, the invention relates to a method of processing an animal meat product or by-product to clear BSE-mediating infectious prion protein therefrom, the method comprising treating the animal meat product or by-product with a protease under sufficient temperature and for sufficient time to destroy the BSE-mediating infectious prion protein therein.
The invention relates in a still further aspect to a method of processing an animal meat product or by-product, including treating the animal meat product or by-product with a protease that is effective for destruction of any
BioResource International, Inc.
Fuierer Marianne
Housel James
Hultquist Steven J.
Winkler Ulrike
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