Methods for large scale production of recombinant DNA-Derived TPA or K2S molecules

• US 20030049729A1
• Filed: 11/14/2001
• Published: 03/13/2003
• Est. Priority Date: 11/14/2000
• Status: Active Grant

• Current Assignee: Boehringer Ingelheim International GmbH (C.H. Boehringer Sohn AG & Co. KG)
• Original Assignee: Boehringer Ingelheim International GmbH (C.H. Boehringer Sohn AG & Co. KG)
• Inventors: Manosroi, Aranya, Goetz, Friedrich, Manosroi, Jiradej, Werner, Rolf-Guenther, Tayapiwatana, Chatchai
• Granted Patent: US 6,955,910 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 9/6424 Serine endopeptidases (3.4.21)
• CPC Class Codes: C12N 9/6459 t-plasminogen activator (3.4.21.68), i.e. tPA
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: C12Y 304/21069 Protein C activated (3.4.21.69)

Abstract

The invention belongs to the field of thrombolysis and of tissue plasminogen activator (tPA) derivative production in prokaryotic cells. The invention relates to methods for the production of a recombinant DNA-derived tPA, a variant therof or a (Kringle 2 Serine) K2S molecule or a variant therof in prokaryotic cells, wherein the tPA or K2S or variant is secreted extracellularly as an active and correctly folded protein, and the prokaryotic cell contains and expresses a vector comprising the DNA coding for the tPA or K2S or variant operably linked to the DNA coding for the signal peptide OmpA. The invention further relates to specific K2S derivatives obtainable by the method. The invention further relates to the DNA molecules and the use of the DNA molecules in the methods.

Claims

1. Method for the production of recombinant DNA-derived tissue plasminogen activator (tPA), a tPA variant, a Kringle 2 Serine protease molecule (K2S) or a K2S variant in prokaryotic cells, wherein said tPA, tPA variant, K2S molecule or K2S variant is secreted extracellularly as an active and correctly folded protein, characterized in that the prokaryotic cell contains and expresses a vector comprising the DNA coding for said tPA, tPA variant, K2S molecule or K2S variant operably linked to the DNA coding for the signal peptide OmpA or a functional derivative thereof.

2. Method according to claim 1, characterised in that said the prokaryotic cell contains and expresses a vector comprising the DNA coding for said tPA, tPA variant, K2S molecule or K2S variant operably linked to the DNA coding for the signal peptide OmpA which is operably linked to the nucleic acid molecule defined by the sequence TCTGAGGGAAACAGTGAC (SEQ ID NO:

1) or a functional derivative thereof.

3. Method according to claim 1 or 2, characterised in that the prokaryotic cell is E. coli.

4. Method according to one of claims 1 to 3, characterised in that the the following steps are carried out:

a) the DNA encoding the tPA, tPA variant, K2S molecule or K2S variant is amplified by PCR;

b) the PCR product is purified;

c) said PCR product is inserted into a vector comprising the DNA coding for OmpA signal peptide and the DNA coding for gpIII in such a way that said PCR product is operably linked upstream to the DNA coding for the OmpA signal sequence and linked downstream to the DNA coding for gpIII of said vector;

d) that a stop codon is inserted between said tPA, tPA variant, K2S molecule or K2S variant and gpIII;

e) said vector is expressed by the prokaryotic cell;

f) the tPA, tPA variant, K2S molecule or K2S variant is purified.

5. Method according to one of claims 1 to 4, characterised in that the vector is a phagemid vector comprising the DNA coding for OmpA signal peptide and the DNA coding for gpIII.

6. Method according to one of claims 1 to 5, characterised in that the vector is the pComb3HSS phagemid.

7. Method according to one of claims 1 to 6, characterised in that the DNA Sequence of OmpA linked upstream to K2S comprises the following sequence or a functional variant thereof or a variant due to the degenerate nucleotide code:

8. Method according to one of claims 1 to 7, characterised in that the DNA Sequence of OmpA comprises the following sequence:

9. Method according to one of claims 1 to 8, characterised in that the DNA Sequence of OmpA consists of the following sequence:

10. Method according to one of claims 1 to 9, characterised in that the DNA of the tPA, tPA variant, K2S molecule or K2S variant is preceeded by a lac promotor and/or a ribosomal binding site.

11. Method according to one of claims 1 to 10, characterised in that the DNA coding for the tPA, tPA variant, K2S molecule or K25 variant is selected from the group of DNA molecules coding for at least 90% of the amino acids 87-527, 174-527, 180-527 or 220-527 of the human tissue plasminogen activator protein.

12. Method according to one of claims 5 to 11, characterised in that the DNA Sequence of K2S comprises the following sequence or a functional variant thereof or a variant due to the degenerate nucleotide code:

13. Method according to one of claims 5 to 12, characterised in that the DNA Sequence of K2S consists of the following sequence:

39. Use according to claim 38, wherein said method is a method according to any one of claims 1 to 13.

14. DNA molecule characterized in that it is coding for:

a) the OmpA protein or a functional derivative therof operably linked to

b) a DNA molecule coding for a polypeptide containing the kringle 2 domain and the serine protease domain of tissue plasminogen activator protein.

15. DNA molecule according to claim 14, characterized in that said DNA sequence comprises the following sequence or a functional variant thereof or a variant due to the degenerate nucleotide code:

16. DNA molecule according to claim 14 or 15, characterized in that said DNA sequence consists of the following sequence:

17. DNA molecule according to one of claims 14 to 16, characterized in that said DNA sequence b) is coding for at least 90% of the amino acids 87-527 of the human tissue plasminogen activator protein.

18. DNA molecule according to one of claims 14 to 17, characterized in that said DNA sequence b) is coding for at least 90% of the amino acids 174-527 of the human tissue plasminogen activator protein.

19. DNA molecule according to any one of claims 14 to 18, characterized in that said DNA sequence b) is coding for at least 90% of the amino acids 180-527 of the human tissue plasminogen activator protein.

20. DNA molecule according to any one of claims 14 to 19, characterized in that said DNA sequence b) is coding for at least 90% of the amino acids 220-527 of the human tissue plasminogen activator protein.

21. DNA molecule according to any one of claims 14 to 20, characterized in that said DNA sequence a) is hybridizing under stringent conditions to the following sequence:

22. DNA molecule according to any one of claims 14 to 21, characterized in that said DNA sequence a) consists of the following sequence:

23. DNA molecule according to any one of claims 14 to 22, characterized in that said DNA sequence b) is hybridizing under stringent conditions to the following sequence:

24. DNA molecule according to any one of claims 14 to 23, characterized in that said DNA sequence b) consists of the following sequence:

31. A vector containing a DNA sequence according to any one of claims 14 to 24.

32. A vector according to claim 31, wherein said DNA sequence is preceeded by a lac promoter and a ribosomal binding site.

33. The vector pComb3HSS containing a DNA according to any one of claims 14 to 24, wherein the expression of the gp III protein is suppressed or inhibited by deleting the DNA molecule encoding said gp III protein or by a stop codon between the gene coding for a a polypeptide containing the kringle 2 domain and the serine protease domain of tissue plasminogen activator protein and the protein III gene.

34. A prokaryotic host cell comprising a DNA molecule according to any one of claims 14 to 24.

35. A prokaryotic host cell comprising a vector according to any one of claims 31 to 33.

36. An E. coli host cell comprising a DNA molecule according to any one of claims 14 to 24.

37. An E. coli host cell comprising a vector according to any one of claims 31 to 33.

38. Use of a DNA molecule according to any one of claims 14 to 24 or of a vector according to any one of claims 31 to 33 or a host cell according to any one of claims 34 to 37 in a method for the production of a polypeptide having the activity of tissue plasminogen activator.

25. Fusion protein of OmpA and K2S, characterised in that it comprises a protein characterized by the following amino acid sequence or a fragment, a functional variant, an allelic variant, a subunit, a chemical derivative or a glycosylation variant therof:

26. Fusion protein of OmpA and K2S according to claim 25, characterised in that it consists of a protein characterized by the following amino acid sequence:

27. K2S protein, characterised in that it comprises a protein defined by the sequence SEGN (SEQ ID NO:

9) and a or a variant or a fragment, a functional variant, an allelic variant, a subunit, a chemical derivative, a fusion protein or a glycosylation variant therof.

28. K2S protein according to claim 27, characterised in that it comprises a protein defined by the sequence SEGNSD (SEQ ID NO:

10) and a or a variant or a fragment, a functional variant, an allelic variant, a subunit, a chemical derivative, a fusion protein or a glycosylation variant therof.

29. K2S protein according to claim 28 or 29, characterised in that it comprises a protein characterized by the following amino acid sequence or a fragment, a functional variant, an allelic variant, a subunit, a chemical derivative or a glycosylation variant therof:

30. K2S according to any one of claims 27 to 30, characterised in that it consists of a protein characterized by the following amino acid sequence: