Claims
- 1. A purified or isolated polypeptide comprising an amino acid sequence selected form the group consisting of
(a) an amino acid sequence at least 90% identical to a fragment of the aspartyl protease amino acid sequence set forth in FIG. 3 (SEQ ID NO: 4); (b) an amino acid sequence at least 90% identical to a fragment of the aspartyl protease amino acid sequence set forth in FIG. 2 (SEQ ID NO: 6); wherein the fragment includes aspartyl protease active site tripeptides DTG and DSG and exhibits aspartyl protease activity involved in processing APP into amyloid beta, wherein substitution differences between the amino acid sequence of the polypeptide and the amino acid sequence of the fragment consist of conservative substitutions, and wherein the polypeptide exhibits aspartyl protease activity involved in processing APP into amyloid beta.
- 2. A purified or isolated polypeptide comprising an amino acid sequence selected form the group consisting of
(c) an amino acid sequence at least 95% identical to a fragment of the aspartyl protease amino acid sequence set forth in FIG. 3 (SEQ ID NO: 4); (d) an amino acid sequence at least 95% identical to a fragment of the aspartyl protease amino acid sequence set forth in FIG. 2 (SEQ ID NO: 6); wherein the fragment includes aspartyl protease active site tripeptides DTG and DSG and exhibits aspartyl protease activity involved in processing APP into amyloid beta, wherein substitution differences between the amino acid sequence of the polypeptide and the amino acid sequence of the fragment consist of conservative substitutions, and wherein the polypeptide exhibits aspartyl protease activity involved in processing APP into amyloid beta.
- 3. A purified or isolated polypeptide according to claim 2 or 3, wherein the polypeptide lacks a transmembrane domain.
- 4. A purified or isolated polypeptide according to clam 2 or 3, wherein the polypeptide further comprises a heterologous peptide tag.
- 5. A method for identifying an agent that decreases the protease activity of an aspartyl protease polypeptide, comprising steps of:
(a) expressing the polypeptide of claims 1-4 by growing a host cell transformed or transfected with a polynucleotide that encodes said polypeptide, under conditions wherein the cell expresses the polypeptide encoded by the polynucleotide, (b) measuring proteolytic activity of the polypeptide in the presence and absence of a test agent, and (c) comparing proteolytic activity of the polypeptide in the presence and absence of the test agent, wherein decreased proteolytic activity in the presence of the test agent identifies the test agent as an agent that decreases the protease activity of the aspartyl protease polypeptide.
- 6. A method for identifying an agent that decreases the protease activity of the aspartyl protease polypeptide comprising steps of:
(a) expressing an aspartyl protease polypeptide by growing a host cell transformed or transfected with a polynucleotide that encodes said polypeptide, under conditions wherein the cell expresses the polypeptide encoded by the polynucleotide, (b) measuring the proteolytic activity of the polypeptide in the presence and absence of a test agent,
wherein the polypeptide comprises an amino acid sequence least 95% identical to a fragment of the Asp2 protein amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6, wherein said fragment is a continuous fragment that includes aspartyl protease active site tripeptides DTG and DSG and exhibits aspartyl protease activity involved in processing APP into amyloid beta, wherein said polypeptide lacks a transmembrane domain, and wherein the polypeptide exhibits aspartyl protease activity involved in processing APP into amyloid beta, and (c) comparing proteolytic activity of the polypeptide expressed by the cell in the presence and absence of the test agent, wherein decreased proteolytic activity in the presence of the test agent identifies the test agent as an agent that decreases the protease activity of the aspartyl protease polypeptide.
- 7. A method according to claim 5 or 6, wherein the proteolytic activity of steps (b) and (c) is proteolytic activity towards an APP substrate.
- 8. A method according to claim 7, wherein the APP substrate comprises an amyloid beta (A-beta) processing site.
- 9. A method according to claim 8, wherein the APP substrate comprises the Swedish mutation (K→N, M→L).
- 10. A method according to claim 8, wherein the APP substrate is a peptide comprising a β-secretase cleavage site that comprises the formula P2-P1-P1′-P2′ (SEQ ID NO: 74), wherein
P2 is an amino acid selected from K and N; P1 is an amino acid selected from M and L; P1′ is the amino acid D; and P2′ is the amino acid A.
- 11. A purified and isolated polynucleotide comprising a nucleotide sequence that encodes the polypeptide of claim 1 or 2.
- 12. A polypeptide comprising the amino acid sequence of a mammalian amyloid protein precursor (APP) or fragment thereof containing an APP cleavage site recognizable by a mammalian β-secretase, and further comprising two lysine residues at the carboxyl terminus of the amino acid sequence of the mammalian APP or APP fragment.
- 13. A polypeptide according to claim 12 comprising the amino acid sequence of a mammalian amyloid protein precursor (APP), and further comprising two lysine residues at the carboxyl terminus of the amino acid sequence of the mammalian amyloid protein precursor.
- 14. A polypeptide according to claim 13, wherein the human APP is selected from the group consisting of: an APP695 isoform, an APP 751 isoform, and an APP770 isoform.
- 15. A polypeptide according to claim 14 wherein the human APP comprises at least one variation selected from the group consisting of a Swedish KM→NL mutation and a London V717→F mutation.
- 16. A polynucleotide encoding the polypeptide of claim 12.
- 17. A purified polynucleotide comprising a nucleotide sequence encoding a hu-Asp1 polypeptide, wherein the polypeptide comprises a fragment of the hu-Asp1 amino acid sequence set forth in SEQ ID NO: 2, and wherein the polypeptide lacks the transmembrane domain amino acids 469-492 of SEQ ID NO: 2 and retains hu-Asp1 α-secretase activity.
- 18. A purified polynucleotide comprising a nucleotide sequence that hybridizes under stringent conditions to the non-coding strand complimentary to SEQ ID NO: 1, wherein the nucleotide sequence encodes a polypeptide having Hu-Asp1 α-secretase activity and wherein the polynucleotide lacks nucleotides encoding a transmembrane domain.
- 19. A polypeptide comprising an amino acid sequence at least 95% identical to a fragment of the amino acid sequence of SEQ ID NO: 2, wherein said polypeptide and said fragment lack a transmemebrane domain and retain hu-Asp1 α-secretase activity.
- 20. A polypeptide comprising an amino acid sequence at least 95% identical to a fragment of the amino acid sequence of SEQ ID NO: 2, wherein said polypeptide and said fragment lack the amino terminal amino acids corresponding to the pro-peptide domain and retain APP processing activity.
Parent Case Info
[0001] The present application is a continuation of U.S. application Ser. No. 09/416,901, filed Oct. 13, 1999 which claims priority benefit of U.S. Provisional Patent Application No. 60/155,493, filed Sep. 23, 1999. The present application also claims priority benefit as a continuation-in-part of U.S. patent application Ser. No. 09/404,133 and PCT/US99/20881, both filed Sep. 23, 1999, both of which in turn claim priority benefit of U.S. Provisional Patent Application No. 60/101,594, filed Sep. 24, 1998. All of these priority applications are hereby incorporated by reference in their entirety.
Provisional Applications (2)
|
Number |
Date |
Country |
|
60155493 |
Sep 1999 |
US |
|
60101594 |
Sep 1998 |
US |
Continuations (1)
|
Number |
Date |
Country |
Parent |
09416901 |
Oct 1999 |
US |
Child |
10652830 |
Aug 2003 |
US |
Continuation in Parts (2)
|
Number |
Date |
Country |
Parent |
09404133 |
Sep 1999 |
US |
Child |
10652830 |
Aug 2003 |
US |
Parent |
PCT/US99/20881 |
Sep 1999 |
US |
Child |
10652830 |
Aug 2003 |
US |