Truncated aggrecanase molecules

Abstract

Truncated aggrecanase proteins and nucleotides sequences encoding them as well as processes for producing them are disclosed. Additionally, aggrecanases with amino acid mutations that lead to increased stability and expression levels in comparison with wild-type or native aggrecanases are also disclosed. Aggrecanases of the invention are especially useful for development of compositions for treatment of diseases such as osteoarthritis. Methods for developing inhibitors of the aggrecanase enzymes and antibodies to the enzymes for treatment of conditions characterized by the degradation of aggrecan are also disclosed.

1 - 30 . (canceled)  
     
     
         31 . An isolated, recombinantly-produced or chemically-synthesized aggrecanase, said aggrecanase being a truncated version of the amino acid sequence of SEQ ID NO:2, said truncated version comprising a truncation of at least 10 amino acid residues from the C-terminus of said amino acid sequence, wherein said at least 10 amino acid residues are located C-terminal to a disintegrin domain of said amino acid sequence.  
     
     
         32 . The aggrecanase of  claim 31 , wherein said at least 10 amino acid residues comprise at least 100 amino acids.  
     
     
         33 . The aggrecanase of  claim 31 , wherein said at least 10 amino acid residues comprise at least 300 amino acids.  
     
     
         34 . The aggrecanase of  claim 31 , wherein said at least 10 amino acid residues comprise a thrombospondin (TSP) domain of said amino acid sequence.  
     
     
         35 . The aggrecanase of  claim 31 , wherein said at least 10 amino acid residues comprise two TSP domains of said amino acid sequence.  
     
     
         36 . A fusion protein consisting of: 
 the aggrecanase of  claim 31;  and    at least one peptide tag.    
     
     
         37 . An isolated, recombinantly-produced or chemically-synthesized aggrecanase, said aggrecanase being a mutated version of the amino acid sequence of SEQ ID NO:2, said mutated version comprising a deletion of at least 10 amino acid residues C-terminal to a disintegrin domain of the amino acid sequence of SEQ ID NO:2, wherein, when aligned, the amino acid sequence of SEQ ID NO:2 and the amino acid sequence of the mutated version are at least 70% identical.  
     
     
         38 . The aggrecanase of  claim 37 , wherein said at least 10 amino acids comprise a TSP domain of said amino acid sequence of SEQ ID NO:2.  
     
     
         39 . The aggrecanase of  claim 37 , wherein said at least 10 amino acids comprise two TSP domains of said amino acid sequence of SEQ ID NO:2.  
     
     
         40 . The aggrecanase of  claim 37 , wherein said at least 10 amino acids comprise at least 100 amino acids.  
     
     
         41 . The aggrecanase of  claim 37 , wherein said at least 10 amino acids comprise at least 300 amino acids.  
     
     
         42 . A fusion protein consisting of: 
 the aggrecanase of  claim 37;  and    at least one peptide tag.    
     
     
         43 . The fusion protein of  claim 42 , wherein said peptide tag is selected from the group consisting of a poly-histidine tag, a FLAG tag, a Herpes simplex glycoprotein D, a beta-galactosidase, a maltose binding protein, a streptavidin tag, and a glutathione S-transferase.  
     
     
         44 . An isolated, recombinantly-produced or chemically-synthesized fusion protein consisting of: 
 an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, and 10;    at least one peptide tag; and    at least one peptide linker connecting the amino acid sequence to the at least one peptide tag.    
     
     
         45 . The fusion protein of  claim 44 , wherein the amino acid sequence is SEQ ID NO:4.  
     
     
         46 . The fusion protein of  claim 44 , wherein the amino acid sequence is SEQ ID NO:6.  
     
     
         47 . The fusion protein of  claim 44 , wherein the amino acid sequence is SEQ ID NO:8.  
     
     
         48 . The fusion protein of  claim 44 , wherein the amino acid sequence is SEQ ID NO:10.  
     
     
         49 . A fusion protein consisting of: 
 the aggrecanase of  claim 31;     at least one peptide tag; and    at least one peptide linker connecting the aggrecanase to the at least one peptide tag.    
     
     
         50 . A fusion protein consisting of: 
 the aggrecanase of  claim 37;     at least one peptide tag; and    at least one peptide linker connecting the aggrecanase to the at least one peptide tag.