Patent

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, wherein the reduced temperature is 16-18° C., 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 nickel nitriloacetic acid (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 7 wherein the reducing agent is dithiothreitol (DTT).

9. The method of claim 1 wherein the HCR protein is selected from the group of the seven C. botulinum serotypes, serotypes A, B, C, D, E, F and G.

10. The method of claim 1 wherein the HCR protein is botulinum neurotoxin type A HCR protein.

11. The method of claim 1 where the culture is exposed to reduced temperature overnight.

12. The method of claim 1 wherein filtration is through a 0.2 μm-2 μm filter.

13. The method of claim 1 additionally comprising the step of exposing the HCR protein to an nitriloacetic acid (NTA) column.