Peptide production

• US 5,322,775 A
• Filed: 11/22/1991
• Issued: 06/21/1994
• Est. Priority Date: 06/30/1986
• Status: Expired due to Term

• Current Assignee: Pharming Intellectual Property B.V. (Pharming Group N.V.)
• Original Assignee: Pharmaceutical Productions, Inc.
• Inventors: Lathe, Richard, Clark, Anthony J.
• Primary Examiner(s): Chambers, Jasemine C.
• Application Number:
  US07/796,917
• Time in Patent Office: 942 Days
• Field of Search: 800/2, 800/DIG. 1, 435/69.1, 435/172.3, 435/320.1, 435/317.1, 435/69.6, 435/69.7, 935/11, 935/111, 935/14, 530/412
• US Class Current: 435/69.1
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/00 Genetically modified animals
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/103 Ovine
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/89 using microinjection

Abstract

A method of producing a proteinaceous compound, involves incorporating a DNA sequence coding for a polypeptide chain of said compound into a gene of a mammal (such as a sheep) coding for a milk whey protein in such a way that the DNA sequence is expressed in the mammary gland of the adult female mammal. The proteinaceous compound may be a (optionally modified) protein such as a blood coagulation factor. The DNA sequence is preferably inserted into the first exon of a gene coding for a whey protein such as beta-lactoglobulin. The proteinaceous compound will generally be recovered from milk of the female mammal, but may (for example if it is an enzyme) be used in situ.

Claims

1. A method of producing a proteinaceous compound, the method comprising the steps of

(a) producing milk in a female transgenic, non-human placental mammal, said mammal containing a transgene construct in its somatic and germ cells, said construct comprising

(i) a DNA sequence encoding a polypeptide chain of said proteinaceous compound;

and

(ii) a β

-lactoglobulin promoter operatively linked to said DNA sequence encoding said polypeptide chain;

said transgene construct being integrated in such a way that said DNA sequence encoding said polypeptide chain is expressed in the mammary gland of said female mammal and said polypeptide is present in said milk; and

(b) collecting the milk produced in step (a).

3. A method as claimed in claim 1 further comprising:

(c) recovering said proteinaceous compound from the collected milk.

4. A method as claimed in claim 1, wherein the proteinaceous compound is a blood coagulation factor or a subunit of a blood coagulation factor.

7. A method as claimed in claim 1 or 2, wherein said transgene construct also comprises a start site for transcription.

8. A method as claimed in claim 1 or 2, wherein said transgene construct also comprises a 3'"'"' sequence flanking a milk protein gene.

9. A method as claimed in claim 1, wherein said transgene construct comprises a signal peptide for the polypeptide chain.

10. A method as claimed in any one of claims 1 or 2, wherein the 3'"'"' end of the DNA sequence terminates after its stop codon, but before its own polyA addition site.

11. A method as claimed in claim 1, wherein said transgene construct additionally comprises one or more of:

(a) a 5'"'"' regulatory sequence (in addition to the promoter) of a β

-lactoglobulin gene;

(b) structural β

-lactoglobulin sequences;

or

(c) the 3'"'"' sequences normally flanking a β

-lactoglobulin gene.

13. A method as claimed in claim 1, wherein the proteinaceous compound is Factor IX.

14. A method as claimed in claim 1, wherein the proteinaceous compound is alpha₁ -antitrypsin.

15. A method as claimed in claim 1, wherein said DNA sequence encoding said polypeptide chain of said proteinaceous compound is cDNA.

16. A method as claimed in claim 9, wherein said signal peptide is a β

-lactoglobulin signal peptide and wherein said signal peptide is fused to said DNA sequence encoding said polypeptide chain of said proteinaceous compound.

2. A method of producing a substance which is the reaction product of an enzyme which catalyzes the formation of said reaction product from one or more substrates of said enzyme present in milk, the method comprising the steps of

(a) producing milk in a female, transgenic, non-human, placental mammal, said mammal containing a transgene construct in its somatic and germ cells, said construct comprising

(i) a DNA sequence encoding said enzyme;

and

(ii) a β

-lactoglobulin promoter operatively linked to said DNA sequence encoding said enzyme;

said transgene construct being integrated in such a way that said DNA sequence encoding said enzyme is expressed in the mammary gland of said female mammal, whereby said reaction product is produced in said milk; and

(b) collecting the milk produced in step (a).

5. A method as claimed in claim 2, wherein the reaction product is recovered from the collected milk.

6. A method as claimed in claim 2, wherein the mammal is a dairy sheep.

12. A method as claimed in claim 2, wherein said transgene construct additionally comprises one or more of:

(a) a 5'"'"' regulatory sequence (in addition to the promoter) of a β

-lactoglobulin gene;

(b) structural β

-lactoglobulin sequences;

or

(c) the 3'"'"' sequences normally flanking a β

-lactoglobulin gene.