Methods and compositions relating to CRM197

Patent metadata

Field	Value
Publication number	US-10287330-B2
Application number	US-201314655801-A
Country	US
Kind code	B2
Filing date	Dec 24, 2013
Priority date	Dec 27, 2012
Publication date	May 14, 2019
Grant date	May 14, 2019

Abstract

The present invention provides novel methods of producing diphtheria toxin. In particular, the present invention provides novel methods of producing nontoxic forms of diphtheria toxin, e.g., CRM197. The present invention also provides novel compositions comprising diphtheria toxin or nontoxic forms of diphtheria toxin, e.g., CRM197.

First claim

What is claimed is:

1. A method of producing CRM197 comprising the steps:
(a) culturing at 18-37 degrees C.° a E. coli cell comprising an expression plasmid containing a nucleic acid encoding CRM197 operatively linked to a promoter;
wherein the nucleic acid encoding CRM197 is fused to a nucleic acid encoding a heterologous signal peptide that targets CRM197 to the periplasm of the E. coli cell;
wherein the nucleic acid encoding a heterologous signal peptide encodes a cleavage site between the signal peptide that targets CRM197 to the periplasm and the CRM197 protein;
wherein the cleavage site sequence comprises the amino acid sequence aa1-aa2-aa3-(cleavage site)-aa4-aa5-aa6-aa7;
wherein
(A) wherein the cleavage site sequence comprises the amino acid sequence aa1-aa2-aa3-(cleavage site)-aa4-aa5-aa6-aa7; and
(B) wherein aa4 to aa7 is selected from ala-asp-asp-val (SEQ ID NO: 7) and ala-gly-ala-asp (SEQ ID NO: 10) and met-gly-ala-asp (SEQ ID NO: 11); and

(b) inducing expression of CRM197 at a culture density of OD600>0.3 at a temperature of 18-37 degrees C.°.

2. The method of claim 1 wherein the wild type signal peptide of CRM197 has been deleted.

3. The method of claim 1 wherein the wild type signal peptide of CRM197 has been replaced by the heterologous signal peptide.

4. The method of claim 1 wherein the heterologous signal peptide is selected from the group consisting of the signal peptide from E. coli outer membrane porin (OmpA), E. coli maltose binding protein (MalE), E. coli DsbA, E. carotovorans pectate lyase (PelB), and Bacillus sp. endoxylanase (XynA).

5. The method of claim 1 wherein CRM197 is produced at a concentration of at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 mg protein per liter culture medium.

6. The method of claim 1 wherein at least 50% of the produced protein is properly folded as determined by circular dichroism.

7. The method of claim 1 wherein at least 50% of the produced protein is not present in aggregates.

8. The method of claim 1 wherein at least 50% of the produced protein is soluble.

9. The method of claim 1 wherein the high copy expression plasmid encodes the sequence of SEQ ID NO: 3 or 5.

10. The method of claim 1 wherein the nucleic acid encoding a heterologous signal peptide is operatively linked to a promoter selected from the group consisting of the 1-arabinose inducible araBAD promoter (PBAD), the lac promoter, the 1-rhamnose inducible rhaP BAD promoter, the T7 RNA polymerase promoter, the trc and tac promoter, the lambda phage promoter p L, and the anhydrotetracycline-inducible tetA promoter/operator.

11. The method of claim 1 wherein the high copy expression plasmid is selected from the group consisting of: pEC415, pBR322, pBAD, pET series, pUC series, pACT3, pEXT22, pEXT20, pBLUESCRIPT series, and pGEM series.

12. The method of claim 1 wherein the expression of CRM197 is induced at a culture density of OD600>1.

13. The method of claim 1 wherein CRM197 is expressed at a temperature of 20, 25, 30, 32, 35° C. or 37° C.

14. The method of claim 1 wherein at least 50% of the expressed CRM197 have an N-terminus of ADDV (SEQ ID NO: 7) or MGADDV (SEQ ID NO: 12).

15. The method of claim 1 wherein at least 50% of the expressed CRM197 have a disulfide bond between Cys186 and Cys201 (SEQ ID NO: 6).

16. The method of claim 5 wherein at least 50% of the produced protein is properly folded as determined by circular dichroism.

17. The method of claim 5 wherein at least 50% of the produced protein is not present in aggregates.

18. The method of claim 5 wherein at least 50% of the produced protein is soluble.

19. The method of claim 5 wherein the high copy expression plasmid encodes the sequence of SEQ ID NO: 3 or 5.

20. The method of claim 5 wherein the nucleic acid encoding a heterologous signal peptide is operatively linked to a promoter selected from the group consisting of the 1-arabinose inducible araBAD promoter (PBAD), the lac promoter, the 1-rhamnose inducible rhaP BAD promoter, the T7 RNA polymerase promoter, the trc and tac promoter, the lambda phage promoter p L, and the anhydrotetracycline-inducible tetA promoter/operator.

21. The method of claim 5 wherein the high copy expression plasmid is selected from the group consisting of: pEC415, pBR322, pBAD, pET series, pUC series, pACT3, pEXT22, pEXT20, pBLUESCRIPT series, and pGEM series.

22. The method of claim 5 wherein the expression of CRM197 is induced at a culture density of OD600>1.

23. The method of claim 5 wherein CRM197 is expressed at a temperature of 20, 25, 30, 32, 35° C. or 37° C.

24. The method of claim 5 wherein at least 50% of the expressed CRM197 have an N-terminus of ADDV (SEQ ID NO: 7) or MGADDV (SEQ ID NO: 12).

25. The method of claim 5 wherein at least 50% of the expressed CRM197 have a disulfide bond between Cys186 and Cys201 (SEQ ID NO: 6).

26. The method of claim 1 , wherein
(A) aa1 is selected from Ala, Ser, Gly, Cys, Thr, and Gln;
(B) aa2 is selected from any natural amino acid; and
(C) aa3 is selected from any natural amino acid except Phe, His, Tyr, Trp, Asp, Glu, Lys, Arg, Asn, and Gln.

27. The method of claim 1 , wherein
(A) aa1 is selected from Ala, Ser, Gly, Cys, Thr, and Gln;
(B) aa2 is Met; and
(C) aa3 is selected from any natural amino acid except Phe, His, Tyr, Trp, Asp, Glu, Lys, Arg, Asn, and Gln.

Assignees

• Glaxosmithkline Biological S A
• Glaxosmithkline Biologicals Sa

Inventors

• Ihssen Julian
• Kowarik Michael
• Thony-Meyer Linda Christiane

Classifications

• C07K14/34 Primary

  from Corynebacterium (G) · CPC title