Production for recombinant lactoferrin and lactoferrin polypeptides using DNA sequences in various organisms

• US 6,100,054 A
• Filed: 05/30/1995
• Issued: 08/08/2000
• Est. Priority Date: 05/05/1989
• Status: Expired due to Term

• Current Assignee: Baylor College of Medicine (Baylor College)
• Original Assignee: Baylor College of Medicine (Baylor College)
• Inventors: Headon, Denis R., O'Malley, Bert W., Conneely, Orla M., May, Gregory S.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Moore, William W.
• Application Number:
  US08/456,108
• Time in Patent Office: 1,897 Days
• Field of Search: 530/350, 530/400, 530/412, 435/69.1, 435/254.11, 435/254.21, 435/254.23, 435/254.3, 435/320.1, 435/69.7, 536/23.5
• US Class Current: 435/69.1
• 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. A recombinant expression vector having a transcription unit comprising the following elements in operable 5'"'"'- to 3'"'"'-linkage:

(1) a promoter permitting the regulation of gene expression in an Aspergillus host cell;

(2) a nucleotide sequence permitting translation initiation in an Aspergillus host cell;

(3) a lactoferrin-encoding nucleotide sequence selected from the group of sequences encoding a naturally-occurring lactoferrin allelic product, an iron-binding lobe of a native lactoferrin, and an antimicrobial peptide of a native lactoferrin having the amino acid sequence between position 20 and position 71, inclusive, of a naturally-occurring lactoferrin precursor; and

,

(4) nucleotide sequences permitting translation and transcription termination in an Aspergillus host cell.

2. The expression vector of claim 1 wherein said promoter is selected from a gene of the group consisting of an alcohol dehydrogenase gene, an argB gene, an α

-amylase gene, a glucoamylase gene and a benA gene.

3. The expression vector of claim 1, wherein said transcription termination sequence is selected from the genes of the group consisting of α

-amylase gene, glucoamylase gene, alcohol dehydrogenase gene and benA gene.

4. A recombinant expression vector comprising the following elements in operable 5'"'"'- to 3'"'"'-linkage:

(1) a promoter permitting the regulation of gene expression in a transformed fungal host cell, said promoter selected from a gene of the group consisting of an alcohol dehydrogenase gene, an argB gene, an α

-amylase gene, a glucoamylase gene and a benA gene;

(2) a nucleotide sequence permitting translation initiation in a transformed fungal host cell;

(3) a lactoferrin-encoding nucleotide sequence selected from the group of sequences consisting of a sequence encoding the lactoferrin protein encoded by SEQ ID NO;

1 and a sequence encoding a naturally-occurring lactoferrin product of a variant human allele;

(4) nucleotide sequences permitting translation and transcription termination in a transformed fungal host cell;

wherein said vector permits the expression of a human lactoferrin in, and subsequent processing by, a transformed fungal host cell.

5. A recombinant expression vector comprising the following elements in operable 5'"'"'- to 3'"'"'-linkage:

(1) a promoter permitting the regulation of gene expression in a transformed fungal host cell;

(2) a nucleotide sequence permitting translation initiation in a transformed fungal host cell;

(3) a lactoferrin-encoding nucleotide sequence selected from the group of sequences consisting of a sequence encoding the lactoferrin protein encoded by SEQ ID NO;

1 and a sequence encoding a naturally-occurring lactoferrin product of a variant human allele; and

,

(4) nucleotide sequences permitting translation and transcription termination in a transformed fungal host cell, wherein said transcription termination sequence is selected from a gene of the group consisting of an alcohol dehydrogenase gene, an α

-amylase gene, a glucoamylase gene and a benA gene;

wherein said vector permits the expression of a human lactoferrin in, and subsequent processing by, a transformed fungal host cell.

6. A transformed fungal host cell comprising a vector permitting the expression of a lactoferrin product in said host cell, said vector comprising a nucleotide sequence encoding a lactoferrin product selected from the group of products consisting of a native lactoferrin protein, an iron-binding lobe of a native lactoferrin, and an antimicrobial peptide of a native lactoferrin having the amino acid sequence between position 20 and position 71, inclusive, of a naturally-occurring lactoferrin precursor, and further comprising transcriptional and translational regulatory elements suitable for the expression of said lactoferrin-encoding nucleotide sequence in operable linkage thereto.

7. The transformed fungal cell of claim 6, wherein said fungal cell is Aspergillus.

8. The transformed Aspergillus cell of claim 6, wherein said Aspergillus cell is selected from the group consisting of A. oryzae, A. niger, A nidulans and A. awamori.

9. The transformed fungal cell of claim 6, wherein said cell is a yeast cell selected from the group consisting of Saccharomyces cerevisiae, Kluveromyces lactis, and Pichia pastoris.

10. A transformed fungal host cell comprising a vector permitting the expression of a bovine lactoferrin product in said host cell, said vector comprising in operable 5'"'"'- to 3'"'"'-linkage:

(1) a promoter permitting the regulation of gene expression in a transformed fungal host cell;

(2) a nucleotide sequence permitting translation initiation in a transformed fungal host cell;

(3) a nucleotide sequence encoding the bovine lactoferrin protein encoded by SEQ ID NO;

3; and

,

(4) nucleotide sequences permitting translation and transcription termination in a transformed fungal host cell.

11. The transformed fungal cell of claim 10, wherein said fungal cell is Aspergillus.

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

13. The transformed fungal cell of claim 10, wherein said cell is a yeast cell selected from the group consisting of Saccharomyces cerevisiae, Kluveromyces lactis, and Pichia pastoris.

14. A process for producing lactoferrin which comprises the following steps:

(a) transforming an Aspergillus cell with an expression vector comprising the following elements in 5'"'"'- to 3'"'"'-linkage;

(1) a promoter permitting the regulation of gene expression in a transformed Aspergillus host cell,

(2) a nucleotide sequence permitting translation initiation in a transformed Aspergillus host cell,

(3) a nucleotide sequence encoding a lactoferrin product selected from the group consisting of a native lactoferrin protein, an iron-binding lobe of a native lactoferrin, and an antimicrobial peptide of a native lactoferrin having the amino acid sequence between position 20 and position 71, inclusive, of a naturally-occurring lactoferrin precursor, and,

(4) nucleotide sequences permitting translation and transcription termination in a transformed Aspergillus host cell; and

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

,

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

15. The process of claim 14, wherein said nucleotide sequence encoding a lactoferrin product is the nucleotide sequence of SEQ ID NO:

1.

16. The process of claim 14, wherein said native lactoferrin protein has the amino acid sequence of SEQ ID NO:

2.

17. The process of claim 14, wherein said Aspergillus cell is selected from the group consisting of A. oryzae, A niger, A. nidulans and A. awamori.

18. The process of claim 14, wherein said promoter is selected from the genes of the group consisting of alcohol dehydrogenase gene, argB gene, α

-amylase gene, glucoamylase gene, and benA gene.

19. The process of claim 14, wherein said transcription termination sequence is selected from the genes of the group consisting of α

-amylase gene, glucoamylase gene, alcohol dehydrogenase gene and benA gene.

20. The process of claim 14, wherein said vector further comprises a nucleotide sequence comprising a selectable marker gene and a nucleotide sequence encoding a secretory signal peptide operably linked to said nucleotide sequence encoding a lactoferrin product.

21. The process of claim 20, wherein said selectable marker gene is selected from the genes of the group consisting of pyr4, pyG, andS, argB and trpC.

22. The process of claim 20, wherein said secretory signal peptide-encoding nucleotide sequence is selected from a gene of the group consisting of an α

-amylase gene, a glucoamylase gene and a lactoferrin gene.

23. A method of isolating a lactoferrin product from a fungal nutrient medium which comprises culturing a transformed Aspergillus oryzae host cell comprising a recombinant vector, wherein said vector comprises the following elements in operable 5'"'"'- to 3'"'"'-linkage:

(1) a promoter permitting the regulation of gene expression in a transformed Aspergillus host cell,

(2) a nucleotide sequence permitting translation initiation in a transformed Aspergillus host cell, and,

(3) optionally, a nucleotide sequence encoding a secretory signal peptide,

(4) a nucleotide coding sequence selected from the group consisting of a cDNA encoding a lactoferrin protein having the amino acid sequence of SEQ ID NO;

2, a cDNA encoding a lactoferrin protein having the amino acid sequence of naturally-occurring allelic lactoferrin variant, a cDNA encoding an iron-binding lobe of a native lactoferrin protein, and a cDNA encoding an antimicrobial peptide of a native lactoferrin protein having an amino acid sequence between position 20 and position 71, inclusive, of a naturally-occurring lactoferrin precursor, and

(5) nucleotide sequences permitting translation and transcription termination in a transformed Aspergillus host cell;

wherein said vector further comprises a selectable marker gene, and culture of said transformed Aspergillus oryzae host cell proceeds in suitable nutrient medium until said lactoferrin product is expressed and secreted into the nutrient medium by said host cell and subsequently isolated therefrom.

24. The method of claim 23, wherein the vector is further defined as containing pyr4 selectable marker gene, promoter from the alpha-amylase gene, transcription termination sequence from the glucoamylase gene, and linker sequence from the alpha-amylase gene.