Recombinant clostridium botulinum neurotoxins

1. A nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 1, wherein the sequence encodes a single-chain BoNT/E1 polypeptide.

2. The sequence of claim 1 , wherein the sequence has a maximum of 160 slow codons.

3. A nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 1, wherein the sequence encodes a single-chain BoNT/E1 polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2.

4. The sequence of claim 3 , wherein the single-chain BoNT/E1 polypeptide comprises one or more of the following amino acids, wherein the amino acid position numbering starts with the N-terminal amino acid residue of the polypeptide and ends with the C-terminal amino acid residue thereof:

glycine at position 177;

serine at position 198;

alanine at position 340;

leucine at position 773;

leucine at position 963;

glutamine at position 964;

alanine at position 967; and

asparagine at position 1195.

5. A nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 1, wherein the sequence encodes a single-chain BoNT/E1 polypeptide and comprises at least 785 synonymous codons when compared to SEQ ID NO: 3.

6. A method for producing a soluble single-chain BoNT/E1 polypeptide, the method comprising expressing the nucleic acid sequence of claim 1 in an E. coli expression system.

7. The method of claim 6 , wherein the soluble single-chain BoNT/E1 polypeptide is expressed in the cytoplasm of the E. coli host cell.

8. The method of claim 6 , wherein the soluble single-chain BoNT/E1 polypeptide is expressed at a level of at least 5 mg/L.

9. The method of claim 6 , further comprising lysis of the E. coli host cell to provide an E. coli host cell homogenate containing the soluble single-chain BoNT/E1 polypeptide.

10. A method for producing a soluble di-chain BoNT/E1 protein, the method comprising:

providing a soluble single-chain BoNT/E1 polypeptide having an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 2;

contacting the polypeptide with trypsin in solution, allowing for the trypsin to cleave the single-chain polypeptide, resulting in a di-chain BoNT/E1 protein; and

separating the soluble BoNT/E1 protein from trypsin by contacting the solution containing soluble BoNT/E1 protein and trypsin with a hydrophobic surface, wherein the soluble BoNT/E1 protein preferentially binds to the hydrophobic surface.

11. The method of claim 10 , wherein the polypeptide comprises one or more of the following amino acids, wherein the amino acid position numbering starts with the N-terminal amino acid residue of the polypeptide and ends with the C-terminal amino acid residue thereof:

glycine at position 177;

serine at position 198;

alanine at position 340;

leucine at position 773;

leucine at position 963;

glutamine at position 964;

alanine at position 967; and

asparagine at position 1195.

12. The method of claim 10 , wherein the soluble single-chain BoNT/E1 polypeptide is provided by expressing a nucleic acid sequence in an E. coli expression system, the nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 1 and encoding a single-chain BoNT/E1 polypeptide.

13. The method of claim 10 , wherein the hydrophobic surface is an inert matrix to which a ligand consisting of aryl or alkyl groups is attached.

14. The method of claim 13 , wherein the hydrophobic surface comprises butyl ligands, phenyl ligands, and/or octyl ligands.

15. An active di-chain BoNT/E1 protein,

wherein the first chain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of positions 1-419 of SEQ ID NO: 2;

wherein the second chain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of positions 423-1252 of SEQ ID NO: 2; and

wherein the first and second chains are joined together by a disulphide bond between cysteine 412 on the first chain and cysteine 426 on the second chain;

wherein the sequences include comprises one or more of the following amino acids, wherein the amino acid position numbering starts with the N-terminal amino acid residue of the polypeptide and ends with the C-terminal amino acid residue thereof:

glycine at position 177;

serine at position 198;

alanine at position 340;

leucine at position 773;

leucine at position 963;

glutamine at position 964;

alanine at position 967; and

asparagine at position 1195.

16. An active di-chain BoNT/E1 protein produced using the method of claim 10 .

17. A composition comprising the active di-chain BoNT/E1 protein of claim 15 , wherein said composition is substantially free of trypsin.

18. The composition of claim 17 , wherein the composition contains less than 10 pg trypsin per 100 ng BoNT/E1 protein.

19. A pharmaceutical composition comprising:

the active di-chain BoNT/E1 protein of claim 15 ;

a surfactant; and

water;

wherein the composition does not comprise a protein stabilizing agent and is substantially free of trypsin.

20. The pharmaceutical composition of claim 19 , further comprising:

sodium chloride;

a buffer to maintain pH between 5.5 and 7.5; and

a disaccharide; and

wherein the water is sterile water.

21. An active di-chain BoNT/E1 protein produced by proteolytic cleavage of the single-chain BoNT/E1 polypeptide produced by the method of claim 6 .