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

• US 5,766,939 A
• Filed: 05/30/1995
• Issued: 06/16/1998
• 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:
  US08/453,703
• Time in Patent Office: 1,113 Days
• Field of Search: 435/69.1, 435/172.3, 435/252.3, 435/254.11, 435/254.21, 435/254.23, 435/254.3, 435/320.1, 530/412, 530/23.5
• US Class Current: 435/320.1
• 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 plasmid suitable for the expression in a transformed prokaryotic host cell of a human lactoferrin, or an iron-binding lobe thereof, said plasmid comprising a DNA sequence encoding a naturally-occurring human lactoferrin protein or an iron-binding lobe thereof and further comprising transcriptional and translational regulatory elements capable of regulating the expression of said lactoferrin-encoding DNA sequence in said transformed host cell.

2. A plasmid comprising:

(a) a DNA sequence encoding an amino acid sequence selected from the group consisting of an amino acid sequence encoded by SEQ ID NO;

1, an amino acid sequence of a naturally-occurring allelic variant of SEQ ID NO;

1, and an amino acid sequence of an iron-binding lobe of a naturally-occurring human lactoferrin and,

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

wherein said plasmid permits the expression of a naturally-occurring human lactoferrin or an iron-binding lobe of a naturally-occurring lactoferrin thereof in a transformed prokaryotic host cell.

3. The plasmid of claim 2, wherein said plasmid permits the expression in transformed prokaryotic host cell of a polypeptide comprising a single iron-binding lobe of a naturally-occurring human lactoferrin.

7. A prokaryotic host cell comprising the plasmid of claim 3.

8. A transformed prokaryotic host cell comprising the plasmid of claim 7.

4. A plasmid comprising:

(a) a DNA sequence encoding a N-terminal lactoferrin antibacterial domain which is encoded by SEQ ID NO;

1 and has the amino acid sequence of SEQ ID NO;

2 from position 20 through position 71, inclusive, or is encoded by a naturally-occurring human lactoferrin variant allele and has the native amino acids from position 20 through 71 of the naturally-occurring precursor lactoferrin, and,

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

wherein said plasmid permits the expression of said N-terminal lactoferrin bactericidal domain in a transformed prokaryotic host cell.

5. A prokaryotic host cell comprising the plasmid of claim 4.

6. The plasmid PT7-7hLF3'"'"'.