Rationally designed heparinases derived from heparinase II

Abstract

Modified heparinases having altered binding specificity and activity are provided. Isolated nucleic acids encoding the same as well as vectors and host cells are provided. Methods for using the modified heparinases are also provided.

1 . A modified heparinase II that has the amino acid sequence of the mature peptide of SEQ ID NO: 2, wherein at least one amino acid residue is substituted and the substitution is of the residue corresponding to position 48, 164, 189, 202, 238, 249, 252, 347, 348, 406, 408, 440, 446, 447, 448, 449, 450, 451, 473, 579 or 682 of SEQ ID NO.: 2.  
     
     
         2 - 16 . (canceled)  
     
     
         17 . A pharmaceutical preparation comprising a sterile formulation of the modified heparinase II of  claim 1  and a pharmaceutically acceptable carrier.  
     
     
         18 . An immobilized substantially pure modified heparinase II comprising: 
 a modified heparinase II as in  claim 1 , and    a solid support membrane, wherein the modified heparinase II is immobilized on the solid support membrane.    
     
     
         19 . A method of specifically cleaving a heparin-like glycosaminoglycan, comprising: 
 contacting a heparin-like glycosaminoglycan with the modified heparinase II of  claim 1 .    
     
     
         20 - 32 . (canceled)  
     
     
         33 . A method of specifically cleaving a heparan sulfate-like glycosaminoglycan, comprising: 
 contacting a heparan sulfate containing fluid with the modified heparinase II of  claim 1 .    
     
     
         34 - 37 . (canceled)  
     
     
         38 . An isolated nucleic acid comprising 
 (a) an isolated nucleic acid encoding the modified heparinase II of  claim 1;     (b) nucleic acids which hybridize under stringent hybridization conditions to the nucleic acid of SEQ ID NO 1 or to the complement of the nucleic acid of SEQ ID NO 1 and which are modified to encode a modified heparinase II as described in  claim 1;  and    (c) nucleic acids that differ from the nucleic acids of (b) in codon sequence due to the degeneracy of the genetic code.    
     
     
         39 . A recombinant host cell including the isolated nucleic acid of  claim 38 .  
     
     
         40 . An expression vector including the isolated nucleic acid of  claim 38.