Expression of processed recombinant lactoferrin and lactoferrin polypeptide fragments from a fusion product in aspergillus

• US 5,955,316 A
• Filed: 08/01/1996
• Issued: 09/21/1999
• Est. Priority Date: 05/05/1989
• Status: Expired due to Fees

• Current Assignee: Agennix, Inc. (Agennix AG)
• Original Assignee: Agennix, Inc. (Agennix AG)
• Inventors: Headon, Denis R., O'Malley, Bert W., Conneely, Orla M.
• Primary Examiner(s): Patterson, Jr., Charles L.
• Assistant Examiner(s): Moore, William W.
• Application Number:
  US08/691,123
• Time in Patent Office: 1,146 Days
• Field of Search: 424/439, 514/6, 435/69.7, 435/172.3, 435/252.3, 435/254.14, 435/254.21, 435/254.23, 435/254.3, 435/320.1, 530/350, 530/390, 530/400, 530/412, 536/23.4, 536/23.5
• US Class Current: 435/69.7
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/79 Transferrins, e.g. lactoferrins, ovotransferrins
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 15/80 for fungi

Abstract

The subject invention provides for the production of lactoferrins and lactoferrin polypeptide fragments using the host cells Aspergillus in combination with novel plasmid constructs. More specifically, the subject invention provides novel vector constructs capable of producing lactoferrins and lactoferrin polypeptide fragments in Aspergillus host cells. More particularly, the subject invention provides for novel plasmid constructs suitable for use with Aspergillus and especially Aspergillus awamori, niger and oryzae host cells, which enables them to produce large amounts of recombinant lactoferrins and lactoferrin polypeptide fragments.

Claims

1. A recombinant expression plasmid vector comprising the following components operably linked 5'"'"' to 3'"'"':

(a) a promoter;

(b) a nucleotide sequence encoding a signal peptide;

(c) a nucleotide sequence encoding an amino-terminal portion of an endogenous secreted Aspergillus polypeptide;

(d) a nucleotide sequence encoding a peptide linker, said peptide linker comprising a fungal peptidase cleavage site; and

(e) a nucleotide sequence encoding a lactoferrin or lactoferrin polypeptide fragment;

wherein said vector permits the expression of a lactoferrin or a lactoferrin polypeptide fragment as a fusion protein and the subsequent production of same as a processed protein.

2. The vector of claim 1, wherein said promoter is selected from genes of the group consisting of alcohol dehydrogenase, α

-amylase, glucoamylase, and benA.

3. The vector of claim 2, wherein said promoter is from the glucoamylase gene.

4. The vector of claim 3, wherein the glucoamylase gene is from A. awamori or A. niger.

5. The vector of claim 2, wherein said promoter is from the α

-amylase gene.

6. The vector of claim 5, wherein the α

-amylase gene is from A. oxyzae.

7. The vector of claim 1, wherein said signal sequence is selected from the genes of the group consisting of glucoamylase and α

-amylase.

8. The vector of claim 7, wherein said signal sequence is from the A. awamori or A. niger glucoamylase gene.

9. The vector of claim 7, wherein said signal sequence is from the A. oryzae α

-amylase gene.

10. The vector of claim 1, wherein said highly expressed endogenous gene is selected from the group consisting of α

-amylase gene and glucoamylase gene.

11. The vector of claim 10, wherein said highly expressed endogenous gene is the A. oryzae α

-amylase gene.

12. The vector of claim 10, wherein said highly expressed endogenouse gene is the A. awamori or A. niger glucoamylase gene.

13. The vector of claim 1, wherein said linker sequence is a peptidase cleavage site.

14. The vector of claim 13, wherein said linker sequence encodes the Kex2 peptidase cleavage site.

15. The vector of claim 1, wherein said lactoferrin or lactoferrin polypeptide fragment is human, bovine or porcine.

16. The vector of claim 1, further comprising a transcription termination sequence and a selectable marker.

17. The vector of claim 16, wherein said transcription termination sequence is selected from the genes of the group consisting of α

-amylase, glucoamylase, alcohol dehydrogenase and benA.

18. The vector of claim 17, wherein said transcription termination sequence is from the A. niger glucoamylase gene.

19. The vector of claim 16, wherein said selectable marker gene is selected from the genes of the group consisting of pyr4, pyrG, amdS, argB, trpC, and phleomycin resistance.

20. The vector of claim 19, wherein said selectable marker is the phleomycin resistance gene.

50. Lactoferrin or lactoferrin polypeptide fragment produced when the vector of claim 1 is used to transform Aspergillus fungal cells.

21. A recombinant expression plasmid vector comprising the following components operably linked from 5'"'"' to 3'"'"':

(a) a promoter from the A. awamori glucoamylase gene;

(b) a a nucleotide sequence encoding a signal peptide from the A. awamori glucoamylase gene;

(c) a nucleotide sequence encoding an amino-terminal portion of the A. awamori glucoamylase gene;

(d) a nucleotide sequence encoding a Kex2 peptidase cleavage site whereby there is an endogenous proteolytic enzyme specific for said nucleotide sequence;

(e) a nucleotide sequence encoding lactoferrin or a lactoferrin polypeptide fragment;

(f) a transcription termination sequence from the A. niger glucoamylase gene; and

(g) a phleomycin resistance selectable marker gene;

wherein said vector permits the expression of a lactoferrin or a lactoferrin polypeptide fragment as a fusion protein and the subsequent production of the same as a processed protein.

22. A recombinant expression plasmid vector comprising the following components operably linked from 5'"'"' to 3'"'"':

(a) a promoter from the A. niger glucoamylase gene;

(b) a nucleotide sequence encoding a signal peptide from the A. niger glucoamylase gene;

(c) a nucleotide sequence encoding an amino-terminal portion of the A. niger glucoamylase gene;

(d) a nucleotide sequence encoding Kex2 peptidase cleavage site whereby there is an endogenous proteolytic enzyme specific for said nucleotide sequence;

(e) a nucleotide sequence encoding a lactoferrin or a lactoferrin polypeptide fragment;

(f) a transcription termination sequence from the A. niger glucoamylase gene; and

(g) a phleomycin resistance selectable marker gene;

wherein said vector permits the expression of a lactoferrin or a lactoferrin polypeptide fragment as a fusion protein and the subsequent production of the same as a processed protein after transformation of said vector into A. niger cells.

51. Lactoferrin or lactoferrin polypeptide fragment produced and processed when the vector of claim 22 is used to transform Aspergillus awamori fungal cells.

23. A recombinant expression plasmid vector comprising the following components operably linked from 5'"'"' to 3'"'"':

(a) a promoter from the A. oryzae α

-amylase gene;

(b) a nucleotide sequence encoding a signal peptide from the A. oryzae α

-amylase gene;

(c) a nucleotide sequence encoding an amino-terminal portion of the A. oryzae α

-amylase gene;

(d) a nucleotide sequence encoding Kex2 peptidase cleavage site whereby there is an endogenous proteolytic enzyme specific for said nucleotide sequence;

(e) a nucleotide sequence encoding a lactoferrin or a lactoferrin polypeptide fragment;

(f) a transcription termination sequence from the A. niger glucoamylase gene; and

(g) α

-phleomycin resistance selectable marker gene;

wherein said vector

permits the expression of a lactoferrin or a lactoferrin polypeptide fragment as a fusion protein and the subsequent production of the same as a processed protein.

52. Lactoferrin or lactoferrin polypeptide fragment produced and processed when the vector of claim 23 is used to transform A. oryzae fungal cells.

24. An expression plasmid vector comprising the following components operably linked from 5'"'"' to 3'"'"':

(a) a promoter from the A. awamori glucoamylase gene;

(b) a nucleotide sequence encoding a signal peptide from the A. awamori glucoarnylase gene;

(c) a nucleotide sequence encoding an amino-terminal portion of the A. awamori glucoamylase gene;

(d) a nucleotide sequence encoding Kex2 peptidase cleavage site

whereby there is an endogenous proteolytic enzyme specific for said nucleotide sequence;

(e) a nucleotide sequence encoding a human lactoferrin or a human lactoferrin polypeptide fragment;

(f) a transcription termination sequence from the A. niger glucoamylase gene; and

(g) a phleomycin resistance selectable marker gene;

wherein said plasmid is used for expressing human lactoferrin or a human lactoferrin polypeptide fragment in Aspergillus fungal cells.

25. The plasmid of claim 24, further defined as having ATCC Accession No. 74290 and designated Awa LF 24-1.

26. Aspergillus awamori fungal cells containing the plasmid of claim 24.

27. A process for producing lactoferrin which comprises culturing a transformed Aspergillus fungal cell comprising a recombinant plasmid, wherein said plasmid comprises the following components operably linked from 5'"'"' to 3'"'"':

(a) a promoter;

(b) a nucleotide sequence encoding a signal peptide;

(c) a nucleotide sequence encoding an amino-terminal portion of an endogenous secreted Aspergillus polypeptide;

(d) a nucleotide sequence encoding a peptide linker, said peptide linker comprising a fungal peptidase cleavage site; and

(e) a nucleotide sequence encoding a lactoferrin or a lactoferrin polypeptide fragment;

wherein said transformed Aspergillus fungal cells are cultured in a suitable nutrient medium until lactoferrin protein or lactoferrin polypeptide fragment is produced as a fusion product and then processed via an endogenous proteolytic enzyme specific for said linker sequence, wherein said processed lactoferrin or lactoferrin polypeptide fragment is secreted into the nutrient medium and isolated therefrom.

28. The process of claim 27, wherein said promoter is selected from genes of the group consisting of alcohol dehydrogenase, α

-amylase, glucoamylase, and benA.

29. The process of claim 28, wherein said promoter is from the glucoamylase gene.

30. The process of claim 29, wherein the glucoamylase gene is from A. awamori or A. niger.

31. The process of claim 28, wherein said promoter is from the α

-amylase gene.

32. The process of claim 31, wherein the α

-amylase gene is from A. oryzae.

33. The process of claim 27, wherein said signal sequence is selected from the genes of the group consisting of glucoamylase and α

-amylase.

34. The process of claim 33, wherein said signal sequence is from the A. awamori glucoamylase gene.

35. The process of claim 33, wherein said signal sequence is from the A. oryzae α

-amylase gene.

36. The process of claim 27, wherein said highly expressed endogenous gene is selected from the group consisting of α

-amylase gene and glucoamylase gene.

37. The process of claim 36, wherein said highly expressed endogenous gene is the A. oryzae α

-amylase gene.

38. The process of claim 36, wherein said highly expressed endogenouse gene is the A. awamori glucoamylase gene.

39. The process of claim 27, wherein said linker sequence is a peptidase cleavage site.

40. The process of claim 38, wherein said linker sequence encodes the Kex2 peptidase cleavage site.

41. The process of claim 27, wherein said plasmid further comprises a transcription termination sequence and a selectable marker gene.

42. The process of claim 41, wherein said transcription termination sequence is selected from the genes of the group consisting of α

-amylase, glucoamylase, alcohol dehydrogenase and benA.

43. The process of claim 42, wherein said transcription termination sequence is from the A. niger glucoamylase gene.

44. The process of claim 28, wherein said selectable marker gene is selected from the genes of the group consisting of pyr4, pyrG, amdS, argB, trpC, and phleomycin resistance.

45. The process of claim 44, wherein said selectable marker is the phleomycin resistance gene.

53. Lactoferrin protein or lactoferrin polypeptide fragment produced by the process of claim 27.

54. The lactoferrin protein of claim 53, wherein the lactoferrin or lactoferrin polypeptide fragment is the human, the porcine or the bovine lactoferrin.

46. A process for producing lactoferrin which comprises culturing a transformed Aspergillus awamori fungal cell comprising a recombinant plasmid, wherein said plasmid comprises the following components operably linked from 5'"'"' to 3'"'"':

(a) a promoter from the A. awamori glucoamylase gene;

(b) a nucleotide sequence encoding a signal peptide from the A. awamori glucoamylase gene;

(c) a nucleotide sequence encoding an amnino-terminal portion of the A. awamori glucoamylase gene;

(d) a nucleotide sequence encoding Kex2 peptidase cleavage site

whereby there is an endogenous proteolytic enzyme specific for said nucleotide sequence;

(e) a nucleotide sequence encoding a human lactoferrin or a human lactoferrin polypeptide fragment;

(f) a transcription termination sequence from the A. niger glucoamylase gene; and

(g) a phleomycin resistance selectable marker gene;

wherein said transformed Aspergillus awamori fungal cell is cultured in a suitable nutrient medium until lactoferrin or lactoferrin polypeptide fragment is produced as a fusion product and then processed via an endogenous proteolytic enzyme specific for said linker sequence, wherein said processed lactoferrin or lactoferrin polypeptide fragment is secreted into the nutrient medium and isolated therefrom.

47. A process for producing lactoferrin which comprises culturing a transformed Aspergillus oryzae fungal cell comprising a recombinant plasmid, wherein said plasmid comprises the following components operably linked from 5'"'"' to 3'"'"':

(a) a promoter from the A. oryzae α

-amylase gene;

(b) a nucleotide sequence encoding a signal peptide from the A. oryzae α

-amylase gene;

(c) a nucleotide sequence encoding an amino-terminal portion of the A. oryzae α

-amylase gene;

(d) a nucleotide sequence encoding Kex2 peptidase cleavage site whereby there is an endogenous proteolytic enzyme specific for said nucleotide sequencer;

(e) a nucleotide sequence encoding a lactoferrin or a lactoferrin polypeptide fragment;

(f) a transcription termination sequence from the A. niger glucoamylase gene; and

(g) α

-phleomycin resistance selectable marker gene;

wherein said transformed Aspergillus oryzae fungal cell is cultured in a suitable nutrient medium until lactoferrin or a lactoferrin polypeptide fragment is produced as a fusion product and then processed via an endogenous proteolytic enzyme specific for said nucleotide sequence, wherein said processed lactoferrin or lactoferrin polypeptide fragment is secreted into the nutrient medium and isolated therefrom.

48. A process for producing lactoferrin which comprises culturing a transformed Aspergillus niger fungal cell comprising a recombinant plasmid, wherein said plasmid comprises the following components operably linked from 5'"'"' to 3'"'"':

(a) a promoter from the A. niger glucoamylase gene;

(b) a nucleotide sequence encoding a signal peppide from the A. niger glucoamylase gene;

(c) a nucleotide sequence encoding an amino-terminal portion of the A. awamori glucoamylase gene;

(d) a nucleotide sequence encoding Kex2 peptidase cleavage site whereby there is an endogenous proteolytic enzyme specific for said nucleotide sequence;

(e) a nucleotide sequence encoding lactoferrin or lactoferrin polypeptide fragment;

(f) a transcription termination sequence from the A. niger glucoamnylase gene; and

(g) a phleomycin resistance selectable marker gene;

wherein said transformed Aspergillus niger fungal cell is cultured in a suitable nutrient medium until lactoferrin or lactoferrin polypeptide fragment is produced as a fusion product and then processed via an endogenous proteolytic enzyme specific for said linker sequence, wherein said processed lactoferrin or lactoferrin polypeptide fragment is secreted into the nutrient medium and isolated therefrom.

49. A method of isolating lactoferrin polypeptide fragment from fungal nutrient medium comprising culturing a transformed Aspergillus awamori fungal cell comprising a recombinant plasmid vector, wherein said plasmid vector comprises a promoter from A. awamori glucoamylase gene, a signal sequence from the A. awamori glucoamylase gene, a 5'"'"' portion of the A. awamori glucoamylase gene, a linker sequence encoding Kex2 peptidase cleavage site whereby there is an endogenous proteolytic enzyme is specific for said linker sequence, a nucleotide sequence encoding a lactoferrin or lactoferrin polypeptide fragment, a transcription termination sequence from the A. niger glucoamylase gene, and a phleomycin resistance selectable marker gene and wherein said transformed Aspergillus awamori fungal cells are cultured in a suitable nutrient medium until lactoferrin or lactoferrin polypeptide fragment is produced as a fusion product and then processed via an endogenous proteolytic enzyme specific for said linker sequence, wherein said processed lactoferrin or lactoferrin polypeptide fragment is secreted into the nutrient medium and isolated therefrom.

55. A lactoferrin protein or a lactoferrin polypeptide fragment as produced by a process for producing lactoferrin which comprises culturing a transformed Aspergillus awamori fungal cell comprising a recombinant plasmid, wherein said plasmid comprises the following components operably linked from 5'"'"' to 3'"'"':

(a) a promoter from the A. awamori glucoamylase gene;

(b) a nucleotide sequence encoding a signal peptide from the A. awamori glucoamylase gene;

(c) a nucleotide sequence encoding an amino-terminal portion of the A. awamori glucoamylase gene;

(d) a nucleotide sequence encoding Kex2 peptidase cleavage site

whereby there is an endogenous proteolytic enzyme specific for said nucleotide sequence;

(e) a nucleotide sequence encoding lactoferrin or lactoferrin polypeptide fragment;

(f) a transcription termination sequence from the A. niger glucoamylase gene; and

(g) a phleomycin resistance selectable marker gene;

wherein said transformed Aspergillus awamori fungal cell is cultured in a suitable nutrient medium until lactoferrin or lactoferrin polypeptide fragment is produced as a fusion product and then processed via an endogenous proteolytic enzyme specific for said linker sequence, wherein said processed lactoferrin or lactoferrin polypeptide fragment is secreted into the nutrient medium and isolated therefrom.