Recombinant clostridium botulinum neurotoxins

Patent metadata

Field	Value
Publication number	US-10030238-B2
Application number	US-201314427234-A
Country	US
Kind code	B2
Filing date	Oct 31, 2013
Priority date	Oct 31, 2012
Publication date	Jul 24, 2018
Grant date	Jul 24, 2018

Abstract

The invention provides a nucleic acid sequence comprising a sequence of contiguous nucleotides, wherein said sequence of contiguous nucleotides has at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 1, and wherein said sequence of contiguous nucleotides encodes a single-chain BoNT/E1 protein. The present invention also provides methods for producing soluble single-chain BoNT/E1 protein in an E. coli host cell, together with methods for producing soluble di-chain BoNT/E1 protein.

First claim

The invention claimed is:

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 .

Assignees

• Ipsen Bioinnovation Ltd
• Ipsen Bioinnovation Ltd
• Ipsen Biopharm Ltd

Inventors

• Cossins Aimee
• Beard Matthew
• Marks Philip

Classifications

• A61K38/4893

  Botulinum neurotoxin (3.4.24.69) · CPC title

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P17/00

  Drugs for dermatological disorders · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• C12N9/52 Primary

  derived from bacteria {or Archaea} · CPC title