Soluble recombinant botulinum toxin proteins

Abstract

The present invention includes recombinant proteins derived from Clostridium botulinum toxins. In particular, soluble recombinant Clostridium botulinum type A, type B and type E toxin proteins are provided. Methods which allow for the isolation of recombinant proteins free of significant endotoxin contamination are provided. The soluble, endotoxin-free recombinant proteins are used as immunogens for the production of vaccines and antitoxins. These vaccines and antitoxins are useful in the treatment of humans and other animals at risk of intoxication with clostridial toxin.

1 . A host cell containing a recombinant expression vector, said vector encoding a protein comprising at least a portion of a  Clostridium botulinum  toxin, said toxin selected from the group consisting of type B toxin and type E toxin.  
     
     
         2 . The host cell of  claim 1 , wherein and said host cell is capable of expressing said protein at a level greater than or equal to 5% of the total cellular protein.  
     
     
         3 . The host cell of  claim 1 , wherein and said host cell is capable of expressing said protein as a soluble protein at a level greater than or equal to 0.25% of the total soluble cellular protein.  
     
     
         4 . The host cell of  claim 1 , wherein said host cell is an  Escherichia coli  cell.  
     
     
         5 . The host cell of  claim 1 , wherein said host cell is an insect cell.  
     
     
         6 . The host cell of  claim 1 , wherein said host cell is a yeast cell.  
     
     
         7 . A host cell containing a recombinant expression vector, said vector encoding a fusion protein comprising a non-toxin protein sequence and at least a portion of a  Clostridium botulinum  toxin, said toxin selected from the group consisting of type B toxin and type E toxin.  
     
     
         8 . The host cell of  claim 7 , wherein said portion of said toxin comprises the receptor binding domain.  
     
     
         9 . The host cell of  claim 7 , wherein said non-toxin protein sequence comprises a poly-histidine tract.  
     
     
         10 . A vaccine comprising a fusion protein, said fusion protein comprising a non-toxin protein sequence and at least a portion of a  Clostridium botulinum  toxin, said toxin selected from the group consisting of type B toxin and type E toxin.  
     
     
         11 . The vaccine of  claim 10  further comprising a fusion protein comprising a non-toxin protein sequence and at least a portion of  Clostridium botulinum  type A toxin.  
     
     
         12 . The vaccine of  claim 10 , wherein said portion of said  Clostridium botulinum  toxin comprises the receptor binding domain.  
     
     
         13 . The vaccine of  claim 10  wherein said non-toxin protein sequence comprises a poly-histidine tract.  
     
     
         14 . The vaccine of  claim 10 , wherein said vaccine is substantially endotoxin-free.  
     
     
         15 . A method of generating antibody directed against a  Clostridium botulinum  toxin comprising: 
 a) providing in any order: 
 i) an antigen comprising a fusion protein comprising a non-toxin protein sequence and at least a portion of a  Clostridium botulinum  toxin, said toxin selected from the group consisting of type B toxin and type E toxin, and  
 ii) a host; and  
   b) immunizing said host with said antigen so as to generate an antibody.    
     
     
         16 . The method of  claim 15 , wherein said antigen further comprises a fusion protein comprising a non-toxin protein sequence and at least a portion of  Clostridium botulinum  type A toxin.  
     
     
         17 . The method of  claim 15 , wherein said portion of said  Clostridium botulinum  toxin comprises the receptor binding domain.  
     
     
         18 . The method of  claim 15  wherein said non-toxin protein sequence comprises a poly-histidine tract.  
     
     
         19 . The method of  claim 15  wherein said host is a mammal.  
     
     
         20 . The method of  claim 19  wherein said mammal is a human.  
     
     
         21 . The method of  claim 15  further comprising step c) collecting said antibodies from said host.  
     
     
         22 . The method of  claim 21  further comprising step d) purifying said antibodies.  
     
     
         23 . The antibody raised according to the method of  claim 15 .  
     
     
         24 . The antibody raised according to the method of  claim 16.