Expression system

Patent metadata

Field	Value
Publication number	US-9994622-B2
Application number	US-201615210245-A
Country	US
Kind code	B2
Filing date	Jul 14, 2016
Priority date	Oct 8, 2009
Publication date	Jun 12, 2018
Grant date	Jun 12, 2018

Abstract

Compositions and methods related to periplasmic expression of a toxin including diphtheria toxin or CRM197 are provided herein.

First claim

The invention claimed is:

1. A process for periplasmic expression of a bacterial toxin, the process comprising growing a culture of Escherichia coli ( E. coli ) host cells containing a polynucleotide comprising:
(a) a 3′ toxin sequence encoding a mature bacterial toxin polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO:32, and
(b) a 5′ signal sequence encoding a signal peptide, wherein the signal peptide directs transport of said bacterial toxin polypeptide to the bacterial periplasm when expressed in said host cell, and wherein the 5′ signal sequence is not derived from Corynebacterium diphtheriae ( C. diphtheriae ); and

inducing expression of said polynucleotide such that said bacterial toxin polypeptide is expressed periplasmically, wherein the process is carried out in a fermentor.

2. The process of claim 1 wherein the 3′ toxin sequence encodes a polypeptide having the amino acid sequence of SEQ ID NO: 32.

3. The process of claim 1 wherein the 3′ toxin sequence comprises SEQ ID NO: 31.

4. The process of claim 1 wherein the 3′ toxin sequence encodes CRM197.

5. The process of claim 1 wherein the 3′ toxin sequence encodes a polypeptide having at least 95% sequence identity to SEQ ID NO: 32.

6. The process of claim 1 wherein the polynucleotide encodes a polypeptide comprising any one of SEQ ID NOs: 33-45.

7. The process of claim 1 wherein the 5′ signal sequence is directly 5′ of the 3′ toxin sequence of the polynucleotide.

8. The process of claim 1 wherein the 5′ signal sequence encodes a signal peptide having an amino acid sequence selected from:
(a) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26;
(b) variants of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 containing 1, 2 or 3 point mutations, insertions or deletions, which variants are capable of directing transport of said bacterial toxin polypeptide to the periplasm of said bacterial host cell, and
(c) fragments of at least 10 amino acids of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, which fragments are capable of directing transport of said bacterial toxin polypeptide to the periplasm of said bacterial host cell.

9. The process of claim 1 wherein the 5′ signal sequence encodes a signal peptide having an amino acid sequence selected from:
(i) SEQ ID NO: 24;
(ii) variants of SEQ ID NO: 24 containing 1, 2 or 3 point mutations, insertions or deletions, which variants are capable of directing transport of said bacterial toxin polypeptide to the periplasm of said bacterial host cell; and
(iii) fragments of at least 10 amino acids of SEQ ID NO: 24, which fragments are capable of directing transport of said bacterial toxin polypeptide to the periplasm of said bacterial host cell.

10. The process of claim 1 wherein the 5′ signal sequence encodes a signal peptide having an amino acid sequence selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26.

11. The process of claim 1 wherein the 5′ signal sequence encodes a signal peptide having an amino acid sequence of SEQ ID NO: 24.

12. The process of claim 1 wherein the 5′ signal sequence comprises a nucleic acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25.

13. The process of claim 1 wherein the 5′ signal sequence comprises SEQ ID NO: 23.

14. A process for periplasmic expression of a bacterial toxin, the process comprising growing a culture of E. coli host cells containing a polynucleotide comprising:
(a) a 3′ toxin sequence encoding a mature bacterial toxin polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO:32, and
(b) a 5′ signal sequence encoding a signal peptide, wherein the signal peptide directs transport of said bacterial toxin polypeptide to the bacterial periplasm when expressed in said host cell, and wherein the 5′ signal sequence is not derived from C. diphtheriae , and wherein the signal peptide has an amino acid sequence selected from:
(i) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26;
(ii) variants of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 containing 1, 2 or 3 point mutations, insertions or deletions, which variants are capable of directing transport of said bacterial toxin polypeptide to the periplasm of said bacterial host cell, and
(iii) fragments of at least 10 amino acids of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, which fragments are capable of directing transport of said bacterial toxin polypeptide to the periplasm of said bacterial host cell, and

inducing expression of said polynucleotide such that said bacterial toxin polypeptide is expressed periplasmically, wherein the process is carried out in a fermentor.

15. The process of claim 14 wherein the 3′ toxin sequence encodes a polypeptide having the amino acid sequence of SEQ ID NO: 32.

16. The process of claim 14 wherein the 3′ toxin sequence comprises SEQ ID NO:31.

17. The process of claim 14 wherein the 3′ toxin sequence encodes CRM197.

18. The process of claim 14 wherein the 3′ toxin sequence encodes a polypeptide having at least 95% sequence identity to SEQ ID NO: 32.

19. The process of claim 14 wherein the polynucleotide encodes a polypeptide comprising any one of SEQ ID NOs: 33-45.

20. The process of claim 14 wherein the 5′ signal sequence is directly 5′ of the 3′ toxin sequence of the polynucleotide.

21. The process of claim 14 , wherein the 5′ signal sequence encodes a signal peptide having an amino acid sequence selected from:
(a) SEQ ID NO: 24;
(b) variants of SEQ ID NO: 24, varying from the corresponding sequence by 1, 2 or 3 point mutations, amino acid insertions or amino acid deletions, which variants are capable of directing transport of said bacterial toxin polypeptide to the periplasm of said bacterial host cell; and
(c) fragments of at least 10 amino acids of SEQ ID NO: 24, which fragments are capable of directing transport of said bacterial toxin polypeptide to the periplasm of said bacterial host cell.

22. The process of claim 14 wherein the 5′ signal sequence encodes a signal peptide having an amino acid sequence selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26.

23. The process of claim 14 wherein the 5′ signal sequence encodes a signal peptide having an amino acid sequence of SEQ ID NO: 24.

24. The process of claim 14 wherein the 5′ signal sequence comprises a nucleic acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25.

25. The process of claim 14 wherein the 5′ signal sequence comprises SEQ ID NO: 23.

26. The process of claim 1 comprising a further step (c) of harvesting cell paste from the culture and purifying the bacterial toxin polypeptide.

27. The process of claim 26 further comprising a step of mixing the purified bacterial toxin polypeptide with a pharmaceutically acceptable excipient.

28. The process of claim 14 comprising a further step (c) of harvesting cell paste from the culture and purifying the bacterial toxin.

29. The process of claim 28 further comprising a step of mixing the purified bacterial toxin polypeptide with a pharmaceutically acceptable excipient.

Assignees

• Glaxosmithkline Biologicals Sa

Inventors

• Blais Normand
• Dehottay Philippe Marc Helene
• Dewerchin Marianne
• Goffin Philippe
• Martin Denis

Classifications

• A61P31/04

  Antibacterial agents · CPC title

• C07K2319/034

  containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved · CPC title

• C12Y204/02036

  NAD(+)--diphthamide ADP-ribosyltransferase (2.4.2.36) · CPC title

• C07K14/34 Primary

  from Corynebacterium (G) · CPC title

• C12N9/1077

  Pentosyltransferases (2.4.2) · CPC title