Rationally designed polysaccharide lyases derived from chondroitinase B

Abstract

The invention relates to rationally designed polysaccharide lyases and uses thereof. In particular, the invention relates to modified chondroitinase B. The modified chondroitinase B enzymes of the invention are useful for a variety of purposes, including cleaving and sequencing polysaccharides such as glycosaminoglycans (GAGs) as well as removing polysaccharides from a solution. The invention also includes methods of inhibiting anticoagulant activity, inhibiting angiogenesis, treating cancer, and inhibiting maternal malarial infection.

1 . A modified chondroitinase B having an amino acid sequence of the mature peptide of SEQ ID NO: 2 or conservative substitutions thereof, wherein at least one residue at a position selected from the group consisting of 116, 184, 213, 219, 245, 250, 271, 272, 296, 298, 318, 333, 363 and 364 of SEQ ID NO: 2 has been substituted or deleted.  
     
     
         2 - 3 . (canceled)  
     
     
         4 . A modified chondroitinase B having a modified product profile, wherein the modified product profile of the modified chondroitinase B is at least 10% different than a native product profile of a native chondroitinase B.  
     
     
         5 - 7 . (canceled)  
     
     
         8 . A modified chondroitinase B having a k cat  or K M  value for a substrate that is at least 10% different than a native chondroitinase B k cat  or K M  value.  
     
     
         9 - 11 . (canceled)  
     
     
         12 . The chondroitinase B of  claim 8 , wherein the substrate is a glycosaminoglycan.  
     
     
         13 . A pharmaceutical preparation comprising a sterile formulation of the chondroitinase B of  claim 1  and a pharmaceutically acceptable carrier.  
     
     
         14 . A method of specifically cleaving chondroitin sulfate, comprising: contacting chondroitin sulfate with the chondroitinase B of  claim 1 .  
     
     
         15 . A method of specifically cleaving dermatan sulfate, comprising: contacting dermatan sulfate with the chondroitinase B of  claim 1 .  
     
     
         16 . The method of  claim 14 , wherein the method is a method of removing chondroitin sulfate from a chondroitin sulfate containing fluid.  
     
     
         17 . The method of claims  15 , wherein the method is a method of removing dermatan sulfate from a dermatan sulfate containing fluid.  
     
     
         18 . The method of  claim 14 , wherein the method is a method for sequencing chondroitin sulfate oligosaccharides.  
     
     
         19 . The method of  claim 15 , wherein the method is a method for sequencing dermatan sulfate oligosaccharides.  
     
     
         20 . A method of analyzing a sample of polysaccharides, comprising: 
 contacting the sample with the chondroitinase B of  claim 1 .    
     
     
         21 . A method of identifying the presence of a particular polysaccharide in a sample, comprising: 
 contacting the sample with the chondroitinase B of  claim 1 .    
     
     
         22 . A method of determining the purity of sample of polysaccharides, comprising: 
 contacting the sample with the chondroitinase B of  claim 1 .    
     
     
         23 . A method for determining the composition of a sample of polysaccharides, comprising: 
 contacting the sample with the chondroitinase B of  claim 1 .    
     
     
         24 . A method for inhibiting angiogenesis, comprising administering to a subject an effective amount for inhibiting angiogenesis of the chondroitinase B or a GAG fragment produced by the chondroitinase B of  claim 1 .  
     
     
         25 . The method of  claim 24 , wherein the chondroitinase B or GAG fragment is administered to a tumor.  
     
     
         26 - 27 . (canceled)  
     
     
         28 . An immobilized modified chondroitinase B comprising: 
 a modified chondroitinase B as in  claim 1 , and    a solid support membrane, wherein the modified chondroitinase B is immobilized on the solid support membrane.    
     
     
         29 . A method for purifying a recombinant chondroitinase, comprising: 
 inducing a culture of cells containing a recombinant chondroitinase with an inducing agent for greater than four hours, and    isolating the recombinant chondroitinase from the cells to produce a purified chondroitinase.    
     
     
         30 - 34 . (canceled)  
     
     
         35 . A method for isolating a recombinant chondroitinase, comprising: 
 lysing a cell culture containing a recombinant chondroitinase having a terminal Histidine tag, and    passing the recombinant chondroitinase over a charged Ni 2+ column to isolate the recombinant chondroitinase.