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Generated: September 18, 2019

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Claims for Patent: 10,077,447

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Summary for Patent: 10,077,447
Title:Coryneform bacterium and method for producing heterologous fusion proteins
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.
Inventor(s): Smirnov; Sergey Vasilievich (Moscow, RU), Kotliarova; Veronika Aleksandrovna (Moscow, RU), Fujii; Hidemi (Kanagawa, JP), Sugiyama; Masakazu (Kanagawa, JP)
Assignee: AJINOMOTO CO., INC. (Tokyo, JP)
Application Number:14/924,201
Patent Claims: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.

28. A method for producing a heterologous fusion protein by secretory production, comprising: cultivating the bacterium of claim 1 in a culture medium; and collecting the heterologous fusion protein produced by secretory production.

29. A method for producing a protein ligated to a substance, comprising: producing a heterologous fusion protein by the method of claim 28, and reacting the heterologous fusion protein with a reactant, wherein the reactant comprises the substance, or the method further comprises modifying the reactant with the substance.

30. The method according to claim 29, wherein the heterologous fusion protein comprises a thioester or an ester bond in between the extein and the intein, and said thioester or said ester bond is cleaved by reacting the heterologous fusion protein with a reactant comprising a nucleophilic group selected from amino group, thiol group and hydroxyl group.

31. The method according to claim 29, wherein the heterologous fusion protein comprises a thioester bond in between the extein and the intein, and said thioester bond is cleaved by reacting the heterologous fusion protein with the reactant comprising thiol group.

32. The method according to claim 31, wherein the heterologous fusion protein is reacted with the reactant in the presence of 2-mercaptoethansulfonic acid.

33. The method according to claim 29, wherein the reactant comprises a toxin.

34. The method according to claim 29, wherein the reactant comprises a drug.

35. The method according to claim 29, wherein the reactant comprises a polyethylene glycol, a radioisotope-labeled compound, or a second polypeptide.

36. A method for producing a peptide or a protein, which is amidated at the C-terminus, comprising: producing a heterologous fusion protein by the method of claim 28, and reacting the heterologous fusion protein with ammonia or a salt thereof.

37. The method according to claim 36, wherein the heterologous fusion protein is reacted with ammonia or a salt thereof, in the presence of a compound containing a nucleophilic thiol group.

Summary for Patent:   Try a Free Trial

Foriegn Application Priority Data
Foreign Country Foreign Patent Number Foreign Patent Date
Russian Federation2013119826Apr 29, 2013

Details for Patent 10,077,447

Applicant Tradename Biologic Ingredient Dosage Form BLA Number Approval Date Patent No. Assignee Estimated Patent Expiration Status Orphan Source
Genentech HERCEPTIN trastuzumab VIAL; INTRAVENOUS 103792 001 1998-09-25   Try a Free Trial AJINOMOTO CO., INC. (Tokyo, JP) 2033-04-29 RX Orphan search
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Number >Approval Date >Patent No. >Assignee >Estimated Patent Expiration >Status >Orphan >Source

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