Coryneform bacterium and method for producing heterologous fusion proteins

Patent metadata

Field	Value
Publication number	US-10077447-B2
Application number	US-201514924201-A
Country	US
Kind code	B2
Filing date	Oct 27, 2015
Priority date	Apr 29, 2013
Publication date	Sep 18, 2018
Grant date	Sep 18, 2018

Abstract

The present invention provides a coryneform bacterium having an ability to produce a heterologous fusion protein by secretory production, which has been modified to express a genetic construct for secretory production of the heterologous fusion protein encoding at least a heterologous fusion protein comprising an extein and an intein having an activity of acyl rearrangement. The method for producing proteins modified at the C-terminus is also provided.

First claim

The invention claimed is:

1. A coryneform bacterium having an ability to produce a heterologous fusion protein by secretory production, wherein said bacterium has been modified to express a genetic construct that allows for secretory production of the heterologous fusion protein, wherein said genetic construct comprises a DNA encoding at least a heterologous fusion protein, wherein said heterologous fusion protein comprises an extein, and an intein having an activity of acyl rearrangement.

2. The coryneform bacterium according to claim 1 , wherein said extein further comprises a target protein.

3. The coryneform bacterium according to claim 2 , wherein said extein further comprises a linker, which is linked to the C-terminus of the target protein and is in between the target protein and the intein.

4. The coryneform bacterium according to claim 3 , wherein said linker comprises a sequence of one or more amino acid residues.

5. The coryneform bacterium according to claim 4 , wherein said linker has a —NH—CH(R1)-CO—NH-CH(R2)-CO— motif at the C-terminus, where R1 and R2 are a side-chain group of a proteinogenic L-amino acid of the same or different kinds.

6. The coryneform bacterium according to claim 5 , wherein said R1 is the side-chain group of any proteinogenic L-amino acid or hydrogen, and R2 is the side-chain group of L-cysteine.

7. The coryneform bacterium according to claim 2 , wherein said target protein is a heterologous protein for the coryneform bacterium.

8. The coryneform bacterium according to claim 7 , wherein said target protein is selected from the group consisting of a bioactive protein, a receptor protein, an antigenic protein, and an enzyme.

9. The coryneform bacterium according to claim 8 , wherein said bioactive protein is selected from the group consisting of a growth factor, a hormone, a cytokine, and an antibody-related molecule.

10. The coryneform bacterium according to claim 8 , wherein said bioactive protein is an exenatide selected from the group consisting of:
(A) a protein having the amino acid sequence of SEQ ID NO: 34, and
(B) a protein having the amino acid sequence of SEQ ID NO: 34, but which includes substitution, deletion, insertion, or addition of one or several amino acid residues, and has an activity of the protein having the amino acid sequence of SEQ ID NO: 34.

11. The coryneform bacterium according to claim 9 , wherein said antibody-related molecule is a protein selected from the group consisting of Fab, F(ab′)2, an Fc-fusion protein, scFv, and combinations thereof.

12. The coryneform bacterium according to claim 11 , wherein said Fab is a trastuzumab Fab having a heavy chain selected from the group consisting of:
(C) a protein having the amino acid sequence of SEQ ID NO: 35, and
(D) a protein having the amino acid sequence of SEQ ID NO: 35, but which includes substitution, deletion, insertion, or addition of one or several amino acid residues and has activity according to the amino acid sequence of SEQ ID NO: 35;
and said trastuzumab Fab has a light chain selected from the group consisting of:
(E) a protein having the amino acid sequence of SEQ ID NO: 36, and
(F) a protein having the amino acid sequence of SEQ ID NO: 36, but which includes substitution, deletion, insertion, or addition of one or several amino acid residues and has activity according to the amino acid sequence of SEQ ID NO: 36.

13. The coryneform bacterium according to claim 10 , wherein said intein is selected from the group consisting of:
(G) an amino acid sequence of SEQ ID NO: 37, and
(H) an amino acid sequence of SEQ ID NO: 37, but which includes substitution, deletion, insertion, or addition of one or several amino acid residues and has activity of acyl rearrangement.

14. The coryneform bacterium according to claim 12 , wherein said intein is selected from the group consisting of:
(I) an amino acid sequence of SEQ ID NO: 38, 40, 41 or 42, and
(J) an amino acid sequence of SEQ ID NO: 38, 40, 41 or 42, but which includes substitution, deletion, insertion, or addition of one or several amino acid residues and has activity of acyl rearrangement.

15. The coryneform bacterium according to claim 12 , wherein said intein is selected from the group consisting of:
(K) an amino acid sequence of SEQ ID NO: 37, and
(L) an amino acid sequence of SEQ ID NO: 37, but which includes substitution, deletion, insertion, or addition of one or several amino acid residues and has activity of acyl rearrangement.

16. The coryneform bacterium according to claim 1 , wherein said bacterium has been modified further so that activity of a penicillin-binding protein is reduced.

17. The coryneform bacterium according to claim 16 , wherein said activity of the penicillin-binding protein is reduced by attenuating expression of a gene encoding the penicillin-binding protein or disrupting the gene.

18. The coryneform bacterium according to claim 17 , wherein said penicillin-binding protein is PBP1a or PBP1b.

19. The coryneform bacterium according to claim 18 , wherein said penicillin-binding protein is selected from the group consisting of:
(M) an amino acid sequence of SEQ ID NO: 44 or 46, and
(N) a protein having the amino acid sequence of SEQ ID NO: 44 or 46, but which includes substitution, deletion, insertion, or addition of one or several amino acid residues and has activity that if the activity thereof is reduced in the coryneform bacterium, amount of the heterologous fusion protein produced by secretory production is increased compared with that observed for a non-modified strain.

20. The coryneform bacterium according to claim 16 , wherein said bacterium has been modified further so that activity of a cell surface layer protein is reduced.

21. The coryneform bacterium according to claim 20 , wherein said activity of the cell surface layer protein is reduced by attenuating expression of a gene encoding the cell surface layer protein or disrupting the gene.

22. The coryneform bacterium according to claim 21 , wherein said cell surface layer protein is selected from the group consisting of PS1, CspB, and CspA.

23. The coryneform bacterium according to claim 22 , wherein said cell surface layer protein is selected from the group consisting of:
(O) an amino acid sequence of SEQ ID NO: 56, 57 or 58, and
(P) a protein having the amino acid sequence of SEQ ID NO: 56, 57 or 58, but which includes substitution, deletion, insertion, or addition of one or several amino acid residues and has activity that if the activity thereof is reduced in the coryneform bacterium, amount of the heterologous fusion protein produced by secretory production is increased compared with that observed for a non-modified strain.

24. The coryneform bacterium according to claim 1 , wherein said genetic construct for secretory production of the heterologous fusion protein further comprises a promoter that functions in the coryneform bacterium.

25. The coryneform bacterium according to claim 1 , wherein said genetic construct for secretory production of the heterologous fusion protein further comprises a signal peptide that functions in the coryneform bacterium.

26. The coryneform bacterium according to claim 1 , wherein said bacterium belongs to the genus Corynebacterium or Brevibacterium.

27. The coryneform bacterium according to claim 26 , wherein said bacterium is Corynebacterium glutamicum.

Assignees

• Ajinomoto Kk

Inventors

• Smirnov Sergey Vasilievich
• Kotliarova Veronika Aleksandrovna
• Fujii Hidemi
• Sugiyama Masakazu

Classifications

• C07K16/32

  against translation products of oncogenes · CPC title

• C07K2317/622

  Single chain antibody (scFv) · CPC title

• C07K2317/55

  Fab or Fab' · CPC title

• C07K2317/54

  F(ab')2 · CPC title

• C07K2317/52

  Constant or Fc region; Isotype · CPC title