Production of recombinant lactoferrin and lactoferrin polypeptides using cDNA sequences in various organisms

• US 5,849,881 A
• Filed: 05/30/1995
• Issued: 12/15/1998
• Est. Priority Date: 05/05/1989
• Status: Expired due to Term

• Current Assignee: Bert W. O'Malley, Denis R. Headon, Gregory S. May, Orla M. Conneely
• Original Assignee: Bert W. O'Malley, Denis R. Headon, Gregory S. May, Orla M. Conneely
• Inventors: O'Malley, Bert W., Conneely, Orla M., May, Gregory S., Headon, Denis R.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Moore, William W.
• Application Number:
  US08/456,106
• Time in Patent Office: 1,295 Days
• Field of Search: 530/350, 530/400, 530/412, 435/69.1, 435/252.11, 435/252.21, 435/252.23, 435/252.3, 435/320.1, 536/23.5
• US Class Current: 530/400
• 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: C12N 15/80 for fungi

Abstract

The verified cDNA sequences for human, bovine and porcine lactoferrin protein have been used to prepare recombinant lactoferrin for therapeutic and nutritional applications. Regions of the cDNA such as the Fe binding sites can be used to make an hLF polypeptide product.

The present invention provides novel plasmids, transfected eucaryotic cells and methods of producing these plasmids and transfected eucaryotic cells. The novel plasmid contains the cDNA for lactoferrin protein. Methods for the production of lactoferrin protein in fungi and bacteria are also provided. Thus, the present invention provides an efficient and economical means for the production of recombinant lactoferrin protein and lactoferrin related polypeptides.

Claims

1. An isolated lactoferrin product produced by the expression of a nucleotide segment having the coding sequence of a naturally-occurring lactoferrin allele in a fungal host cell transformed with said lactoferrin-encoding nucleotide sequence, wherein said lactoferrin-encoding nucleotide sequence is a cDNA and said cDNA is operably linked to a promoter element utilized by said fungal host cell.

2. The isolated lactoferrin product of claim 1 wherein said transformed fungal host cell is a yeast cell.

3. The isolated lactoferrin product of claim 2, wherein said transformed yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces lactis, and Pichia pastoris.

4. The isolated lactoferrin product of claim 1 wherein said transformed fungal cell is Aspergillus.

5. The isolated lactoferrin product of claim 4, wherein said transformed Aspergillus cell is selected from the group consisting of A. oryzae, A. niger, A. nidulans and A. awamori.

6. The isolated lactoferrin product of claim 1 or claim 2, wherein said lactoferrin product has a glycosylation pattern that is different from that of a naturally-occurring lactoferrin.

7. The isolated lactoferrin product of claim 1 wherein said cDNA encodes a polypeptide having the amino acid sequence of SEQ ID NO:

2.

8. The isolated lactoferrin product of claim 2 wherein said cDNA encodes a polypeptide having the amino acid sequence of SEQ ID NO:

2.

9. The isolated lactoferrin product of claim 7 or claim 8, wherein said lactoferrin product has a glycosylation pattern that is different from that of a naturally-occurring lactoferrin.

10. The isolated lactoferrin product of claim 7, wherein said transformed fungal cell is Aspergillus.

11. The isolated lactoferrin product of claim 10, wherein said transformed Aspergillus cell is selected from the group consisting of A. oryzae, A. niger, A. nidulans and A. awamori.

12. The isolated lactoferrin product of claim 8, wherein said transformed yeast cell is selected from the group consisting of Saccharormyces cerevisiae, Kluveromyces lactis, and Pichia pastoris.

13. An isolated lactoferrin product produced by the expression of a nucleotide segment having the coding sequence of a naturally-occurring lactoferrin allele in a fungal host cell transformed with said lactoferrin-encoding nucleotide sequence, wherein said lactoferrin-encoding nucleotide sequence is a cDNA and said cDNA is operably linked to a promoter element utilized by said fungal host cell, wherein said lactoferrin is deglycosylated after expression.

14. The isolated lactoferrin product of claim 13 wherein said transformed fungal host cell is a yeast cell.

15. The isolated lactoferrin product of claim 14, wherein said transformed yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Kluveromyces lactis, and Pichia pastosis.

16. The isolated lactoferrin product of claim 13, wherein said transformed fungal cell is Aspergillus.

17. The isolated lactoferrin product of claim 16, wherein said transformed Aspergillus cell is selected from the group consisting of A. oryzae, A. niger, A. nidulans and A. awamori.

18. An isolated lactoferrin fragment produced by the expression of a nucleotide segment encoding a polypeptide having the amino acid sequence of SEQ ID NO:

2 from position 20 through position 71, inclusive, in a fungal host cell transformed with said lactoferrin fragment-encoding nucleotide sequence, wherein said lactoferrin fragment-encoding nucleotide sequence is a cDNA sequence and said cDNA sequence is operably linked to a promoter element utilized by said fungal host cell.

19. The isolated lactoferrin fragment of claim 18, wherein said transformed host cell is a yeast cell.

20. The isolated lactoferrin fragment of claim 18, wherein said transformed yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Kluveromyces lactis, and Pichia pastoris.

21. The isolated lactoferrin fragment of claim 18, wherein said transformed fungal cell is Aspergillus.

22. The isolated lactoferrin fragment of claim 21, wherein said transformed Aspergillus cell is selected from the group consisting of A. oryzae, A. niger, A. nidulans and A. awamori.

23. An isolated lactoferrin iron-binding lobe produced by the expression of a nucleotide sequence encoding a lactoferrin iron-binding lobe comprising a single iron-binding site of a naturally-occurring lactoferrin allele in a fungal host cell transformed with said lactoferrin iron-binding lobe-encoding nucleotide sequence, wherein said nucleotide coding nucleotide sequence is a cDNA sequence and said cDNA sequence is operably linked to a promoter element utilized by said fungal host cell.

24. The isolated lactoferrin iron-binding lobe of claim 23, wherein said cDNA is endogenous to a human source.

25. The isolated lactoferrin iron-binding lobe of claim 23 or claim 24 wherein said transformed fungal host cell is a yeast cell.

26. The isolated lactoferrin iron-binding lobe of claim 25, wherein said transformed yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Kluveromyces lactis, and Pichia pastoris.

27. The isolated lactoferrin iron-binding lobe of claim 23 or claim 24 wherein said transformed fungal host cell is Aspergillus.

28. The isolated lactoferrin iron-binding lobe of claim 27, wherein said transformed Aspergillus cell is selected from the group consisting of A. oryzae, A. niger, A. nidulans and A. awamori.

29. A human lactoferrin protein manufactured by a recombinant process of production comprising the following steps:

(a) transforming a fungal host cell with an expression plasmid comprising,

(i) a DNA sequence of a naturally-occurring human lactoferrin allele which encodes the amino acid sequence encoded by SEQ ID NO;

1, and,

(ii) a promoter, a translation initiation sequence, and transcription and translation termination sequences,

wherein said plasmid is suitable for the expression of said human lactoferrin in said transformed host cell; and

,

(b) culturing said transformed host cell in a suitable nutrient medium until said human lactoferrin is expressed and secreted into the nutrient medium; and

,

(c) isolating said secreted human lactoferrin from the nutrient medium.

30. A deglycosylated lactoferrin protein manufactured by a recombinant process of production comprising the following steps:

(a) transforming an Aspergillus host cell with an expression vector comprising,

(i) a DNA sequence encoding a naturally-occurring lactoferrin protein and,

(ii) a promoter and a transcription termination sequence,

wherein said vector is suitable for the expression of said human lactoferrin in said transformed Aspergillus host cell;

(b) culturing said transformed Aspergillus host cell in a suitable nutrient medium until said lactoferrin is expressed and secreted into the nutrient medium;

(c) isolating said secreted lactoferrin from the nutrient medium; and

(d) deglycosylating said lactoferrin product.

31. The lactoferrin protein of claim 30, wherein said lactoferrin product is human.

32. A human lactoferrin protein fragment manufactured by a recombinant process of production comprising the following steps:

(a) transforming an Aspergillus host cell with an expression vector comprising,

(i) a DNA sequence encoding a human lactoferrin protein fragment having the amino acid sequence of SEQ ID NO;

2 between positions 20 and 71, inclusive, and

(ii) a promoter, a translation initiation sequence, and transcription and translation termination sequences,

wherein said vector is suitable for the expression of said human lactoferrin fragment in said transformed eukaryotic host cell;

(b) culturing said transformed eukaryotic host cell in a suitable nutrient medium until said human lactoferrin fragment is expressed and secreted into the nutrient medium; and

,

(c) isolating said secreted human lactoferrin fragment from the nutrient medium.

33. A lactoferrin protein manufactured by a recombinant process comprising the following steps:

(a) transforming an Aspergillus host cell with an expression vector comprising,

(i) a DNA sequence encoding a naturally-occurring lactoferrin protein and,

(ii) a promoter and a transcription termination sequence,

wherein said vector is suitable for the expression of said human lactoferrin in said transformed Aspergillus host cell;

(b) culturing said transformed Aspergillus host cell in a suitable nutrient medium until said lactoferrin is expressed and secreted into the nutrient medium; and

(c) isolating said secreted lactoferrin from the nutrient medium.