Patent EA-005163-B1 (EA)
Title: Methods for large scale production of recombinant dna-derived tpa or k2s molecules
Inventor: WERNER ROLF-GUNTER
IPC: A61K38/46
Filed: 2001-11-07
Abstract: 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 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 preceded 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 K2S 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: 14. DNA molecule characterized in that it is coding for: a) the OmpA protein or a functional derivative thereof 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 b) 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 b) consists of the following sequence: 23. Fusion protein of OmpA and K2S, characterised in that it comprises a protein characterized by the following amino acid sequence: 24. Fusion protein of OmpA and K2S according to claim 23, characterised in that it consists of a protein characterized by the following amino acid sequence: 25. K2S protein, characterised in that it comprises a protein characterized by the following amino acid sequence: 26. A vector containing a DNA sequence according to any one of claims 14 to 22. 27. A vector according to claim 26, wherein said DNA sequence is preceded by a lac promotor and a ribosomal binding site. 28. 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 polypeptide containing the kringle 2 domain and the serine protease domain of tissue plasminogen activator protein and the protein III gene. 29. A prokaryotic host cell comprising a DNA molecule according to any one of claims 14 to 22. 30. A prokaryotic host cell comprising a vector according to any one of claims 26 to 28. 31. An E. coli host cell comprising a DNA molecule according to any one of claims 14 to 22. 32. An E. coli host cell comprising a vector according to any one of claims 26 to 28. 33. Use of a DNA molecule according to any one of claims 14 to 22 or of a vector according to any one of claims 26 to 28 or a host cell according to any one of claims 29 to 32 in a method for the production of a polypeptide having the activity of tissue plasminogen activator. 34. Use according to claim 33, wherein said method is a method according to any one of claims 1 to 13.