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

• US 5,571,691 A
• Filed: 10/28/1993
• Issued: 11/05/1996
• 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:
  US08/145,681
• Time in Patent Office: 1,104 Days
• Field of Search: 536/23.5, 435/240.2, 435/320.1, 435/69.1, 435/255, 435/254, 435/252.3, 530/350, 530/395, 530/394, 530/324
• 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: C07K 2319/02 containing a signal sequence
• 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 DNA sequence encoding human lactoferrin protein, a substitution analog wherein said DNA sequence is a substitution analog of SEQ ID NO:

1 or of a naturally-occurring allele of SEQ ID NO;

1.

2. A portion of the DNA sequence of claim 1, further defined as a region encoding a lactoferrin fragment comprising a human lactoferrin iron non-binding site.

3. A DNA sequence encoding human lactoferrin protein, wherein said DNA sequence is SEQ. ID NO:

1, or a naturally-occurring allele of SEQ ID NO;

1.

4. A portion of the DNA sequence of claim 3, further defined as a region encoding a lactoferrin fragment comprising a human lactoferrin iron non-binding site.

5. The DNA sequence of claim 4, further defined as a region encoding a carboxy terminal iron binding site, wherein said carboxy terminal iron binding site is obtained by treating said DNA SEQ ID No. 1 with Sma I and Hind III restriction enzymes.

6. A plasmid adapted for the expression of human lactoferrin in a eukaryotic cell, wherein said plasmid comprises DNA encoding a human lactoferrin protein and regulatory elements necessary for the expression of said DNA in said cell.

11. A eukaryotic cell comprising the plasmid of claim 6.

12. The eukaryotic cell of claim 11, wherein said cell is selected from the group consisting of mammalian and insect cells.

13. The eukaryotic cell of claim 12, wherein said insect cell is SF9.

7. A plasmid comprising:

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

2; and

(b) a promoter, and transcription and translation initiation and termination sequences;

wherein said plasmid is adapted for the expression of human lactoferrin in a eukaryotic cell.

14. A transformed eukaryotic cell comprising the plasmid of claim 7.

8. A plasmid comprising:

(a) DNA as defined by SEQ ID No. 1 or a naturally-occurring allele thereof; and

(b) a promoter, and transcription and translation initiation and termination sequences;

wherein said plasmid is adapted for the expression of human lactoferrin in a eukaryotic cell.

15. A transformed eukaryotic cell comprising the plasmid of claim 8.

9. A recombinant expression plasmid vector having a transcriptional unit comprising:

(a) a promoter;

(b) DNA encoding the amino acid sequence of SEQ ID NO;

2; and

(c) transcription and translation initiation and termination sequences;

wherein said vector permits the expression of a processed form of a human lactoferrin by a transformed host cell.

10. A recombinant expression plasmid vector comprising:

(a) a promoter;

(b) DNA encoding a human lactoferrin as defined by SEQ ID No. 1 or a naturally-occurring allele thereof; and

(c) transcription and translation initiation and termination sequences;

wherein said vector permits the expression of a processed form of a human lactoferrin by a transformed host cell.

16. A method for producing human lactoferrin which comprises the following steps:

(a) transforming a eukaryotic cell with an expression plasmid comprising an expression plasmid vector containing;

i) DNA encoding human lactoferrin having the sequence of SEQ ID No;

1 or a naturally-occurring allele; and

ii) a promoter, and transcription and translation initiation and termination sequences;

wherein said plasmid is adapted for the expression of human lactoferrin in a eukaryotic cell; and

(b) culturing said transformed eukaryotic cell in a suitable nutrient medium until human lactoferrin protein is formed, secreted into the nutrient medium, and isolated therefrom.