Escherichia coli-derived vaccine and therapy against botulism
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.