Claims
- 1. A polypeptide consisting essentially of SEQ ID NO: 4 or SEQ ID NO: 4 having a conservative amino acid substitution.
- 2. A chimeric protein comprising the polypeptide of claim 1.
- 3. A polypeptide comprising a modified ADAM33 catalytic domain, wherein said modified ADAM33 catalytic domain has at least 95% identity with the amino acid sequence of SEQ ID NO: 14; and wherein said polypeptide catalyzes the proteolytic cleavage of a peptide comprising the amino acid sequence of SEQ ID NO: 35.
- 4. The polypeptide of claim 3 wherein the modified ADAM33 catalytic domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16.
- 5. A chimeric protein comprising the polypeptide of claim 4.
- 6. The chimeric protein of claim 5 consisting of the amino acid sequence of SEQ ID NO: 38.
- 7. A nucleic acid having of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33 and SEQ ID NO: 37; wherein the nucleotide sequence can further comprise a heterologous nucleotide sequence.
- 8. The nucleic acid of claim 7 wherein the nucleic acid encodes a polypeptide comprising an ADAM33 pro domain and an ADAM33 catalytic domain;
has a nucleotide sequence selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 31, and SEQ ID NO: 33; and wherein the heterologous nucleotide sequence encodes a secretion signaling sequence having the amino acid sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
- 9. The nucleic acid of claim 8 wherein the heterologous nucleotide sequence further encodes the amino acid sequence of SEQ ID NO: 36.
- 10. An expression vector, comprising the nucleic acid of claim 9, and a transcriptional control sequence, wherein the nucleic acid is operatively linked to the transcriptional control sequence.
- 11. A host cell that comprises the expression vector of claim 10.
- 12. A method for producing a polypeptide comprising an ADAM33 catalytic domain comprising culturing the host cell of claim 11 in a culture medium, wherein the host cell expresses the nucleic acid encoding the polypeptide; and whereby the polypeptide is produced.
- 13. The method of claim 12 wherein the host cell is from a Drosophila melanogaster Schneider 2 (S2) stable cell line.
- 14. The method of claim 13 wherein the transcriptional control sequence comprises a Drosophila metallothionein promoter (PMT); and wherein 1 to 25 μM Cd2+ and 10 μM to 1 mM Zn2+ are added to the host cell to induce expression and to maximize the production of the polypeptide comprising the ADAM33 catalytic domain that is free of the ADAM33 pro domain.
- 15. A method of obtaining a purified form of the polypeptide comprising the ADAM33 catalytic domain that is free of the ADAM33 pro domain comprising purifying the polypeptide produced by the method of claim 14 from the culture medium.
- 16. The purified form of the polypeptide comprising the ADAM33 catalytic domain free of the ADAM33 pro domain obtained by the method of claim 15.
- 17. A method for producing a polypeptide comprising a catalytic domain of a zinc metalloprotease that is free of its pro domain comprising culturing a eukaryotic host cell in a culture medium,
wherein the host cell comprises an expression vector that comprises a nucleic acid that encodes a pro domain and a catalytic domain of the zinc metalloprotease; wherein the nucleic acid is operatively linked to the transcriptional control sequence that comprises a metallothionein promoter; wherein 1 to 25 μM Cd2+ and 10 μM to 1 mM Zn2+ are added to the host cell to induce expression and to maximize the production of the polypeptide comprising the catalytic domain that is free of its pro domain; and wherein when the host cell expresses the nucleic acid, the polypeptide is produced, and the production of the polypeptide comprising the catalytic domain of the zinc metalloprotease that is free of its pro domain is maximized.
- 18. The method of claim 17 wherein the eukaryotic host cell is a Drosophila melanogaster Schneider 2 (S2) cell and the metallothionein promoter is a Drosophila metallothionein promoter.
- 19. A method of obtaining a purified form of a polypeptide comprising the catalytic domain of a zinc metalloprotease that is free of its pro domain comprising purifying the polypeptide produced by the method of claim 18 from the culture medium.
- 20. The purified form of the polypeptide comprising the catalytic domain of the zinc metalloprotease that is free of its pro domain obtained by the method of claim 19.
- 21. A method of treating a subject that has a respiratory disorder comprising administering a therapeutic amount of the recombinant polypeptide of claim 1 to the subject.
- 22. The method of claim 21 wherein said administering is performed by either intramuscular or subcutaneous injection or infusion of the recombinant polypeptide into the subject.
- 23. The method of claim 21 wherein said administering is performed by introducing a vector into the subject as part of a gene therapy protocol, wherein the vector encodes said recombinant polypeptide; and wherein the recombinant polypeptide is expressed in the therapeutic amount in the subject.
Parent Case Info
[0001] This application claims the benefit of U.S. Provisional Applications 60/434,830, filed Dec. 19, 2002, 60/434,802, filed Dec. 19, 2002, and 60/440,263, filed Jan. 15, 2003. These applications are hereby incorporated by reference in their entirety.
Provisional Applications (3)
|
Number |
Date |
Country |
|
60434802 |
Dec 2002 |
US |
|
60434830 |
Dec 2002 |
US |
|
60440263 |
Jan 2003 |
US |