Proteins with Improved Solubility and Methods for Producing and Using Same

Abstract

A method is provided for improving the solubility of proteins, for example, bacterial toxins. In one embodiment, solubility is improved by introducing point mutations that replace cysteine residues capable of forming intermolecular disulfide bonds with other amino acid residues that do not form such bonds. By abrogating the ability of the cysteine residues to form inter-molecular disulfide bonds, aggregation of the protein is reduced, thereby improving the solubility of the protein. In another embodiment, solubility of the protein is improved by producing truncated forms of the protein that express the LHN domain and a fragment of the Hc domain. Proteins made according to the method of the invention are useful, for example, as immunodiagnostic agents and vaccine components.

1 - 34 . (canceled) 
     
     
         35 . A recombinant protein comprising a truncated botulinum serotype E toxin, wherein the truncation improves the solubility of the recombinant protein. 
     
     
         36 . The protein of  claim 35 , wherein the truncation is in the Hc domain. 
     
     
         37 . The protein of  claim 35 , wherein the truncated protein comprises the LH N /E domain and the amino terminal 103 amino acids of the Hc domain. 
     
     
         38 . The protein of  claim 35 , wherein the truncated protein comprises the amino terminal 948 amino acids of the serotype E toxin. 
     
     
         39 . The protein of  claim 35 , wherein the truncated protein comprises the LH N /E domain and the amino terminal 202 amino acids of the Hc domain. 
     
     
         40 . The protein of  claim 35 , wherein the truncated protein comprises the amino terminal 1047 amino acids of the serotype E toxin. 
     
     
         41 . The protein of  claim 35 , wherein the truncated protein comprises the LH N /E domain and the amino terminal 304 amino acids of the Hc domain. 
     
     
         42 . The protein of  claim 35 , wherein the truncated protein comprises the amino terminal 1149 amino acids of the serotype E toxin. 
     
     
         43 . A nucleic acid encoding a recombinant protein of  claim 35 . 
     
     
         44 . A method for improving the solubility of a clostridial neurotoxin, comprising:
 (a) providing a nucleic acid sequence encoding a clostridial neurotoxin;   (b) modifying the nucleic acid sequence so that it encodes the LH N  fragment and a portion of the H c  fragment of the neurotoxin;   (c) transforming the modified nucleic acid sequence into a host cell capable of expressing the modified nucleic acid sequence; and   (d) expressing the modified nucleic acid sequence to produce the protein.   
     
     
         45 . A method of treating or preventing botulism comprising administering a protein of  claim 35  to a patient in need thereof. 
     
     
         46 . A composition comprising a protein of  claim 35  and a pharmaceutically acceptable carrier. 
     
     
         47 . A method of protecting an individual from botulism, comprising administering to the individual a composition of  claim 46 . 
     
     
         48 . A method of producing antibodies that neutralize a clostridial neurotoxin, comprising administering the composition of  claim 46  to an animal, allowing the animal to develop neutralizing antibodies to the clostridial neurotoxin, and isolating an antiserum that neutralizes the clostridial neurotoxin from the animal. 
     
     
         49 . An antiserum produced by the method of  claim 48 . 
     
     
         50 . A method of treating exposure to a clostridial neurotoxin, comprising administering to a patient that has been exposed to the clostridial neurotoxin the antiserum of  claim 49 . 
     
     
         51 . A mutated botulinum serotype E toxin comprising either or both of a leucine residue substituted for the tryptophan residue at position 1223 and a phenylalanine residue for the tyrosine residue at position 1224 of SEQ ID NO: 1 or SEQ ID NO: 2. 
     
     
         52 . A method of treating or preventing botulism comprising administering a protein of  claim 51  to a patient in need thereof. 
     
     
         53 - 58 . (canceled)