Amyloidin protease and uses thereof
AssigneesAthena Neurosciences, Inc., Eli Lilly and Company
InventorsHarry F. Dovey, Peter A. Seubert, Sukanto Sinha
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Abstract
A proteolytic enzyme isolated from human tissue which exhibits a proteolytic activity to hydrolyze Met-Asp peptide bond in an amyloid-like substrate is disclosed. This enzyme has been designated "amyloidin" because it proteolytically cleaves a Met-Asp bond similar to the one present in the amyloid precursor protein to release a fragment having the mature Asp terminus of the .beta.-amyloid peptide. Antibodies to the amyloidin protease are also provided. Methods to isolate and purify the amyloidin protease are provided, as well as assays to screen for inhibitors of the amyloidin protease. Also disclosed is the gene encoding the protease and methods for expression of the protease by recombinant DNA means.
Claims15
Human amyloidin protease, a metalloprotease capable of cleaving the Met-Asp bond in the substrate:
- acetyl-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg (Seq ID No:1), substantially free of natural contaminants, which has a molecular weight of about 80,000 daltons as determined by SDS-polyacrylamide gel electrophoresis.
A method for purifying a human amyloid metalloprotease of approximately 80,000 daltons from cells, said method comprising:
- a) disrupting human cells to form an aqueous extract and an insoluble fraction;
- b) using chromatographic fractionation on the aqueous extract to produce an enriched fraction having amyloidin metalloprotease activity;
- c) binding the enriched fraction of (b) to a hydrophobic interaction chromatography matrix;
- d) eluting the bound metalloprotease activity by gradient fractionation to form an eluate;
- and
- e) selecting fractions from the eluate having the ability to hydrolyze an internal Met-Asp peptide bond in an amyloid-like peptide substrate.
Dependencies are inferred from the claim text.