Claims
- 1. An isolated bovine TNF-α convertase characterized by:
(a) an amino acid sequence comprising a sequence defined by SEQ ID NO: 2, (b) an apparent molecular weight in SDS-PAGE of about 65 kDa, and (c) an ability to cleave human proTNF-α to produce soluble, mature human TNF-α.
- 2. An antibody which specifically binds to the bovine TNF-α convertase of claim 1.
- 3. The antibody of claim 2 which is a monoclonal antibody.
- 4. An isolated or recombinant nucleic acid encoding the bovine TNF-α convertase of claim 1.
- 5. A recombinant vector comprising the nucleic acid of claim 4.
- 6. A host cell comprising the recombinant vector of claim 5.
- 7. A method for making a TNF-α convertase comprising culturing a host cell of claim 6 under conditions in which the nucleic acid is expressed.
- 8. The method of claim 7 in which the convertase is isolated from the culture.
- 9. A method for identifying an inhibitor of a mammalian TNF-α convertase, comprising:
(a) contacting a mammalian TNF-α convertase in the presence of substrate with a sample to be tested for the presence of an inhibitor of the convertase; and (b) measuring the rate of cleavage of the substrate; whereby an inhibitor of the TNF-α convertase in the sample is identified by measuring substantially reduced cleavage of the substrate, compared to what would be measured in the absence of such inhibitor.
- 10. The method of claim 9 which further comprises:
(a) contacting a matrix-degrading metalloprotease in the presence of substrate with an inhibitor of a TNF-α convertase; and (b) measuring the rate of cleavage of the substrate; whereby a specific inhibitor of a TNFα convertase is identified by measuring substantially undiminished cleavage of the substrate, compared to what would be measured in the absence of such inhibitor.
- 11. The method of claim 10 in which the matrix-degrading metalloprotease is selected from the group consisting of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9 and MMP-10.
- 12. The method of claim 9 in which said contacting occurs on the surface of a mammalian host cell comprising one or more nucleic acids encoding a mammalian protein which produces TNF-α convertase activity, and a substrate of the convertase.
- 13. The method of claim 10 in which said contacting occurs on the surface of a mammalian host cell comprising one or more nucleic acids encoding a matrix-degrading metalloprotease and a substrate of the protease.
- 14. The method of claim 12 in which the host cell is from human embryonic kidney cell line 293 or a clone thereof.
- 15. The method of claim 12 in which the substrate is human proTNF-α.
- 16. The method of claim 12 in which the mammalian protein is selected from the group consisting of bovine ADAM 10; human ADAM 10; human matrix-type metalloproteases MT-MMP1, MT-MMP2 and MT-MMP3; and a protein comprising an amino acid sequence defined by SEQ ID NO: 21.
- 17. A method for identifying a nucleic acid encoding a mammalian TNF-α convertase, comprising:
(a) culturing a mammalian host cell comprising a first recombinant expression vector comprising a nucleic acid encoding a TNF-α convertase substrate and a second recombinant expression vector comprising a nucleic acid that is to be tested to determine whether it encodes a mammalian TNF-α convertase, under conditions in which expression occurs; and (b) measuring the rate of cleavage of the substrate; whereby a nucleic acid encoding a mammalian TNF-α convertase is identified by measuring substantially increased cleavage of the substrate, compared to what would be measured in the absence of such nucleic acid.
- 18. The method of claim 17 in which the host cell is a cell from human embryonic kidney cell line 293 or a clone thereof.
- 19. The method of claim 17 in which the substrate is human proTNF-α.
- 20. An isolated protein comprising an amino acid sequence defined by SEQ ID NO: 21.
- 21. An isolated or recombinant nucleic acid encoding the protein of claim 20.
- 22. The nucleic acid of claim 21 which comprises a nucleotide sequence defined by SEQ ID NO: 21.
- 23. A recombinant vector comprising a nucleic acid of claim 21.
- 24. A host cell comprising a recombinant vector of claim 23.
- 25. A method for making a protein comprising culturing a host cell of claim 24 under conditions in which the nucleic acid is expressed.
- 26. The method of claim 25 in which the protein is isolated from the culture.
Parent Case Info
[0001] This application claims the benefit of U.S. Provisional Application 60/021,710, filed Jul. 12, 1996.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60021710 |
Jul 1996 |
US |
Divisions (2)
|
Number |
Date |
Country |
Parent |
09156163 |
Sep 1998 |
US |
Child |
09982308 |
Oct 2001 |
US |
Parent |
08889909 |
Jul 1997 |
US |
Child |
09156163 |
Sep 1998 |
US |
Continuations (1)
|
Number |
Date |
Country |
Parent |
09982308 |
Oct 2001 |
US |
Child |
10145014 |
May 2002 |
US |