Escherichia coli-derived vaccine and therapy against botulism

Abstract

A method of producing botulinum toxin C-terminal receptor binding domain (HCR) is disclosed. The one embodiment, the method comprises the steps of (a) preparing E. coli transformed with an expression vector comprising DNA encoding HCR protein, (b) inducing expression of the HCR protein at a reduced temperature in a culture media, and (c) purifying the HCR protein via extraction, wherein the extraction comprises a clarification by centrifugation and a filtration, wherein the purified HCR protein is at least 10 mg/L of culture medium.

1 . A method of producing  botulinum  toxin C-terminal receptor binding domain (HCR), comprising the steps of 
 (a) preparing  E. coli  transformed with an expression vector comprising DNA encoding at least 95% of the HCR protein,    (b) inducing expression of the HCR protein at a reduced temperature in a culture media, and    (c) purifying the HCR protein via extraction, wherein the extraction comprises a clarification by centrifugation and a filtration, wherein the purified HCR protein is at least 10 mg/L of culture medium.    
     
     
         2 . The method of  claim 1  wherein the protein is at least 15 mg/L of culture medium.  
     
     
         3 . The method of  claim 1  wherein the  E. coli  strain is  E. coli  BL-21 RIL.  
     
     
         4 . The method of  claim 1  wherein the expression vector is a pET expression vector resulting in an N-terminal HIS 6 -HCR/A fusion protein.  
     
     
         5 . The method of  claim 1  wherein the purification of the HCR protein additionally comprises chromatography purification through NI-NTA resin.  
     
     
         6 . The method of  claim 1  wherein the step (c) filtration is through a 0.2-2.0 μm filter.  
     
     
         7 . The method of  claim 1  wherein the purification of the HCR protein is in the presence of a reducing agent.  
     
     
         8 . The method of  claim 4  wherein the reducing agent is DTT.  
     
     
         9 . The method of  claim 1  wherein the HCR protein is selected from the group of the seven  C. botulinum  serotypes and subtypes with the serotypes.  
     
     
         10 . The method of  claim 1  wherein the HCR protein is  botulinum  neurotoxin type A HCR protein.  
     
     
         11 . The method of  claim 9  wherein the HCR domain is selected from  botulinum  neurotoxin types A, B, C, D, E, F and G.  
     
     
         12 . The method of  claim 1  wherein the reduced temperature is 16-18° C.  
     
     
         13 . The method of  claim 1  where the culture is exposed to reduced temperature overnight.  
     
     
         14 . The method of  claim 1  wherein filtration is through a .2 μm-2 μm filter.  
     
     
         15 . The method of  claim 1  additionally comprising the step of exposing the HCR protein to an NTA column.  
     
     
         16 . A HCR protein preparation, prepared by the method of  claim 1 .  
     
     
         17 . An  E. coli  culture medium extraction, wherein the concentration of HCR protein is greater than 10 mg/L.  
     
     
         18 . The extraction of  claim 17 , wherein the concentration is greater than 15 mg/L.