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

• US 6,228,614 B1
• Filed: 03/09/1999
• Issued: 05/08/2001
• Est. Priority Date: 05/05/1989
• Status: Expired due to Fees

• 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): Achutamurthy, Ponnathapu
• Assistant Examiner(s): Moore, William W.
• Application Number:
  US09/265,577
• Time in Patent Office: 791 Days
• Field of Search: 435/69.1, 435/252.3, 435/320.1, 536/23.5, 530/400
• US Class Current: 435/69.1
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/79 Transferrins, e.g. lactoferrins, ovotransferrins

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 method for expressing human lactoferrin in an eukaryotic cell comprising the step of introducing a plasmid comprising DNA encoding a human lactoferrin protein and regulatory elements necessary for the expression of said DNA in said cell.

2. The method of claim 1 wherein the plasmid comprises:

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 permits the expression of human lactoferrin in an eukaryotic cell.

3. The method of claim 1 wherein the plasmid comprises:

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

2 wherein from one to five amino adds are substituted by a different amino acid and wherein said human lactoferrin reversibly binds ferric iron with high affinity; and

,

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

wherein said plasmid permits the expression of human lactoferrin in an eukaryotic cell.

4. The method of claim 1 wherein the plasmid comprises:

a) DNA as defined by sequence of SEQ ID NO;

1; and

,

b) a promoter and transcription and translation initiation and termination sequences.

5. The method of claim 1 wherein the plasmid comprises:

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

2 wherein from one to five amino acids are substituted by a different amino acid and wherein said human lactoferrin reversibly binds ferric iron with high affinity; and

,

b) a promoter and transcription and translation initiation and termination sequences.

6. The method of claim 1 wherein the plasmid has 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.

7. The method of claim 1 wherein the plasmid has a transcriptional unit comprising:

a) a promoter;

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

2 wherein from one to five amino acids are substituted by a different amino acid and wherein said human lactoferrin reversibly binds ferric iron with high affinity; 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.

8. The method of claim 1 wherein the plasmid comprises:

a) a promoter;

b) DNA encoding a human lactoferrin as defined by SEQ ID NO;

1; and

,

c) transcription and translation initiation and termination sequences.

9. A method for producing human lactoferrin which comprises transforming an eukaryotic cell with an expression vector comprising an expression plasmid comprising:

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

1; and

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

wherein said vector permits the expression of human lactoferrin in an eukaryotic cell.

10. A method for producing human lactoferrin which comprises transforming an eukaryotic cell with an expression vector comprising an expression plasmid having:

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

2 wherein from one to five amino acids are substituted by a different amino acid and wherein said human lactoferrin reversibly binds ferric iron with high affinity; and

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

wherein said vector permits the expression of human lactoferrin in an eukaryotic cell.