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1. WO2002024863 - AVIAN AND REPTILE DERIVED POLYNUCLEOTIDE ENCODING A POLYPEPTIDE HAVING HEPARANASE ACTIVITY

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[ EN ]

WHAT IS CLAIMED IS:

1. An isolated nucleic acid comprising a genomic, complementary or composite polynucleotide sequence encoding a polypeptide being at least 75 % similar to SEQ ID NO:4 or a portion thereof as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 8 and length weight equals 2, average match equals 2.912 and average mismatch equals -2.003.

2. The isolated nucleic acid of claim 1, wherein said polypeptide has heparanase catalytic activity or said polypeptide is cleavable by a protease so as to have said heparanase catalytic activity.

3. An isolated nucleic acid comprising a genomic, complementary or composite polynucleotide sequence being at lest 65 % identical to SEQ ID NO: 10 or a portion thereof as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 50, length weight equals 3, average match equals 10 and average mismatch equals -9.

4. The isolated nucleic acid of claim 3, wherein said polynucleotide encodes a polypeptide which has heparanase catalytic activity or which is cleavable by a protease so as to have said heparanase catalytic activity.

5. An isolate nucleic acid as set forth in SEQ ID NO: 10 or a portion thereof.

6. An isolated nucleic acid comprising a genomic, complementary or composite polynucleotide sequence being hybridizable with SEQ ID NO: 10 or a portion thereof under hybridization conditions of hybridization solution containing 10 % dextrane sulfate, 1 M NaCl, 1 % SDS and 5 x 106 cpm 32p labeled probe, at 65 °C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 65 °C

7. The isolated nucleic acid of claim 6, wherein said polynucleotide encodes a polypeptide which has heparanase catalytic activity or which is cleavable by a protease so as to have said heparanase catalytic activity.

8. A nucleic acid construct comprising the isolated nucleic acid of claim 1.

9. A nucleic acid construct comprising the isolated nucleic acid of claim 3.

10. A nucleic acid construct comprising the isolated nucleic acid of claim 4.

11. A nucleic acid construct comprising the isolated nucleic acid of claim 6.

12. A cell transformed or transfected with the nucleic acid of claim 1.

13. A cell transformed or transfected with the nucleic acid of claim 3.

14. A cell transformed or transfected with the nucleic acid of claim 4.

15. A cell transformed or transfected with the nucleic acid of claim 6.

16. An oligonucleotide of at least 17 bases specifically hybridizable with the isolated nucleic acid of claim 3 and which is not hybridizable with any mammalian heparanase cDNA.

17. A pair of oligonucleotides each of at least 17 bases specifically hybridizable with the isolated nucleic acid of claim 3 in an opposite orientation so as to direct exponential amplification of a portion thereof in a nucleic acid amplification reaction, and which are not hybridizable with any mammalian heparanase cDNA..

18. A nucleic acid amplification product obtained using the pair of primers of claim 17.

19. A recombinant protein comprising a polypeptide being at least 75 % similar to SEQ ID NO:4 or a portion thereof as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 8 and length weight equals 2, average match equals 2.912 and average mismatch equals -2.003.

20. The recombinant protein of claim 19, wherein said polypeptide has heparanase catalytic activity or said polypeptide is cleavable by a protease so as to have said heparanase catalytic activity.

21. A recombinant protein comprising a polypeptide being encoded by a nucleic acid including a genomic, complementary or composite polynucleotide sequence being at lest 65 % identical to SEQ ID NO:10 or a portion thereof as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 50, length weight equals 3, average match equals 10 and average mismatch equals -9.

22. The recombinant protein of claim 21, wherein said polypeptide has heparanase catalytic activity or said polypeptide is cleavable by a protease so as to have said heparanase catalytic activity.

23. A recombinant protein comprising a polypeptide being encoded by a nucleic acid as set forth in SEQ ID NO: 10 or a portion thereof.

24. A recombinant protein comprising a polypeptide being encoded by a nucleic acid including a genomic, complementary or composite polynucleotide sequence being hybridizable with SEQ ID NO: 10 or a portion thereof under hybridization conditions of hybridization solution containing 10 % dextrane sulfate, 1 M NaCl, 1 % SDS and 5 x 10^ cpm 3 p labeled probe, at 65 °C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 65 °C.

25. The recombinant protein of claim 24, wherein said polypeptide has heparanase catalytic activity or said polypeptide is cleavable by a protease so as to have said heparanase catalytic activity.

26. A pharmaceutical composition comprising, as an active ingredient, the recombinant protein of claim 19 and a pharmaceutically acceptable carrier.

27. A pharmaceutical composition comprising, as an active ingredient, the recombinant protein of claim 21 and a pharmaceutically acceptable carrier.

28. A pharmaceutical composition comprising, as an active ingredient, the recombinant protein of claim 23 and a pharmaceutically acceptable carrier.

29. A pharmaceutical composition comprising, as an active ingredient, the recombinant protein of claim 24 and a pharmaceutically acceptable carrier.

30. A medical equipment comprising a medical device containing, as an active ingredient, a recombinant protein having heparanase catalytic activity.

31. A nucleic acid construct comprising a first polynucleotide encoding a peptide as set forth at positions 1 to 19 of SEQ ID NO:4 and an in frame, second polynucleotide encoding a membrane targeted or secreted polypeptide.

32. The nucleic acid construct of claim 31, wherein said membrane targeted or secreted polypeptide is human heparanase.

33. A nucleic acid construct comprising a first polynucleotide encoding a signal peptide of avian or reptile heparanase and an in frame, second polynucleotide encoding a membrane targeted or secreted polypeptide.

34. The nucleic acid construct of claim 33, wherein said membrane targeted or secreted polypeptide is human heparanase.

35. A method of expressing a protein of interest in a cell, the method comprising:
transforming the cell with a nucleic acid construct that comprises a first polynucleotide encoding a signal peptide of avian or reptile heparanase and an in frame, second polynucleotide encoding a membrane targeted or secreted polypeptide; and
culturing the cell.