Composition and method for monitoring in vitro conversion of full -length mammalian prion protein to amyloid form with physical properties of PRPsc
OwnerIlia Baskakovinventor
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Abstract
The present invention relates to an automated in vitro method for converting a prion protein into multiple forms including β-oligomer or amyloid forms while monitoring the mechanism and progress of the molecular conversion.
Claims16
An in vitro method for converting a prion protein to an amyloid form, the method comprising:
- a) providing a conversion solution comprising guanidine hydrochloride (GdnHCl);
- b) adding a recombinant full-length prion protein with an intact S-S bind to the conversion solution;
- c) maintaining the pH in the solution in a range from about 5.5 to about 7.0;
- d) exposing the recombinant prion protein to the solution under essentially continuance shaking for a sufficient time to form an amyloid structure
An in vitro method for converting a prion protein to a β-oligomer form, the method comprising:
- a) providing a conversion solution comprising guanidine hydrochloride (GdnHCl);
- b) adding a recombinant full-length prion protein or fragment thereof to the conversion solution;
- c) maintaining the pH in the solution in a range from about 3.0 to about 4.0;
- d) exposing the recombinant prion proteins to the solution for a sufficient time to form an β-oligomer form.
An automated in vitro method of monitoring conversion kinetics of the conversion of a full-length prior protein or fragments thereof, the method comprising:
- a) providing a conversion solution comprising guanidine hydrochloride (GdnHCl) and Thioflavin T (ThT);
- b) adding a full-length prion protein or fragment thereof to the conversion solution;
- c) maintaining the pH in the solution in a range from about 3.0 to about 6.5;
- and
- d) monitoring the conversion kinetics by measuring the fluorescence intensity corresponding to the conversion.
An automated in vitro method for determining test compounds that inhibit or reduce the conversion of a full-length prior protein or fragments thereof into an amyloid or β-oligomer, the method comprising:
- a) providing a conversion solution comprising guanidine hydrochloride (GdnHCl) and Thioflavin T (ThT);
- b) adding a full-length prion protein or fragment thereof to the conversion solution;
- c) maintaining the pH in the solution in a range from about 3.0 to about 7.0;
- d) introducing the test compound;
- and
- e) monitoring the conversion kinetics relative to a control sample without the test compound by measuring the fluorescence intensity corresponding to the conversion.
A kit for determining test compounds that inhibit or reduce the conversion of a full-length prior protein or fragments thereof into a β-oligomer or amyloid form, the kit comprising:
- a) a conversion solution comprising guanidine hydrochloride (GdnHCl) and Thioflavin T (ThT);
- b) a pH altering compound for maintaining the conversion in a range from about 3.0 to about 7.0, wherein a full-length prion protein and test compound are added to the conversion solution and monitoring conditions to determine if the test compound inhibits or reduces conversion.
Dependencies are inferred from the claim text.