Gene encoding chondroitinase ABC and uses therefor

Abstract

Nucleic acid sequences coding for the chondroitinase ABC gene and isolated chondroitinase ABE protein produced in a host cell transformed with a nucleic acid vector directing the expression of a nucleotide sequence coding for chondroitinase ABE protein described. Chondroitinase ABC prepared by chemical synthesis also described. Monoclonal and polyclonal antibodies which are specifically reactive with chondroitinase ABC protein are disclosed. The isolated chondroitinase ABC can be used in methods of treating intervertebral disc replacement, promoting neurite regeneration, and detecting galactosaminoglycans.

1. An isolated nucleic acid fragment encoding chondroitinase ABC, comprising the nucleotide sequence of SEQ ID NO: 1. 
     
     
       2. An expression vector comprising the nucleic acid as defined in  claim 1  operably linked to a regulatory sequence. 
     
     
       3. A host cell transformed with the expression vector as defined in  claim 2 . 
     
     
       4. A host cell of claim  3  23 wherein the cell is eukaryotic. 
     
     
       5. A host cell of claim  3  23 wherein the cell is prokaryotic. 
     
     
       6. A method of producing chondroitinase ABC protein comprising: culturing the host cell as defined in  claim 2  under conditions appropriate for expression; and isolating chondroitinase ABC protein from the culture. 
     
     
       7. An isolated nucleic acid encoding chondroitinase ABC comprising a nucleotide sequence which differs from the nucleotide sequence of SEQ ID NO: 1, due to degeneracy in the genetic code. 
     
     
       8. An expression vector comprising the nucleic acid as defined in  claim 7  operably linked to a regulatory sequence. 
     
     
       9. A host cell transformed with the expression vector as defined in  claim 8 . 
     
     
       10. A method of producing chondroitinase ABC protein comprising:
 culturing the host cell as in defined in  claim 9  under conditions appropriate for expression; and isolating chondroitinase ABC protein from the culture.   
     
     
       11. An isolated nucleic acid fragment comprising the coding region of chondroitinase ABC and having a nucleotide sequence consisting of nucleotides 297-3288 73 to 3066 of SEQ ID NO: 1. 
     
     
       12. An expression vector comprising the nucleic acid of  claim 11  operably linked to a regulatory sequence. 
     
     
       13. A host cell transformed with the expression vector of  claim 12 . 
     
     
       14. A method of producing chondroitinase ABC protein comprising: culturing the host cell as in defined in  claim 13  under conditions appropriate for expression; and isolating chondroitinase ABC protein from the culture. 
     
     
       15. An isolated nucleic acid comprising a nucleotide sequence which differs from nucleotides 297-3288 73 to 3066 of SEQ ID NO: 1, due to degeneracy in the genetic code. 
     
     
       16. An expression vector comprising the nucleic acid as defined in  claim 15  operably linked to a regulatory sequence. 
     
     
       17. An isolated nucleic acid fragment comprising nucleotides 2160-3288 of SEQ ID NO: 1. 
     
     
       18. An expression vector comprising the nucleic acid as defined in  claim 17  operably linked to a regulatory sequence. 
     
     
       19. An isolated nucleic acid comprising a nucleotide sequence which differs from nucleotides 2160-3288 of SEQ ID NO: 1, due to degeneracy in the genetic code. 
     
     
       20. An expression vector comprising the nucleic acid as defined in claims operably linked to a regulatory sequence. 
     
     
       21. An isolated nucleic acid fragment comprising the coding region of the nucleotide sequence of SEQ ID NO: 1. 
     
     
       22. An expression vector comprising the nucleic acid as defined in  claim 21  operably linked to a regulatory sequence. 
     
     
       23. A host cell transformed with the expression vector as defined in  claim 22 . 
     
     
       24. A method of producing chondroitinase ABC protein comprising:
 culturing the host cell as in defined in  claim 23  under conditions appropriate for expression; and isolating chondroitinase ABC protein from the culture. 
 
     
     
       25. An isolated nucleic acid comprising a nucleotide sequence which differs from the coding region of SEQ ID NO: 1, due to degeneracy in the genetic code. 
     
     
       26. An expression vector comprising the nucleic acid as defined in  claim 25  operably linked to a regulatory sequence. 
     
     
       27. A host cell transformed with the expression vector as defined in  claim 26 . 
     
     
       28. A method of producing chondroitinase ABC protein comprising: culturing the host cell as in defined in  claim 27  under conditions appropriate for expression; and isolating chondroitinase ABC protein from the culture. 
     
     
       29. An isolated nucleic acid fragment encoding chondroitinase ABC, wherein the nucleic acid comprises a nucleotide sequence of the insert of pCHS6 obtained from E. coli XL1-Blue/pCHS6 deposited at Accession NO. FERM BP-4170 .