Peptide production

Abstract

A method of producing a substance comprising a peptide, involves incorporating a DNA sequence coding for the peptide 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 substance may be an (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 substance will generally be recovered from milk of the female mammal, but may (for example if it is an enzyme) be used in situ.

1 . A method of producing a substance comprising a polypeptide, the method comprising incorporating a DNA sequence coding for the polypeptide into a gene of a female mammal 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.  
     
     
         2 . A method as claimed in  claim 1 , wherein the substance is subsequently recovered from milk of the adult female mammal.  
     
     
         3 . A method as claimed in  claim 1 , wherein the substance is recovered from the milk after post-transcriptional modification.  
     
     
         4 . A method as claimed in  claim 1 , wherein the substance comprising a peptide is a protein.  
     
     
         5 . A method as claimed in  claim 1 , wherein the substance comprising a polypeptide is a human blood protein.  
     
     
         6 . A method as claimed in  claim 1 , wherein the substance comprising a polypeptide is a peptide hormone.  
     
     
         7 . A method as claimed in  claim 1 , wherein the substance comprising a polypeptide is a blood coagulation factor or a subunit of a blood coagulation factor.  
     
     
         8 . A method as claimed in  claim 1 , wherein the substance comprising a polypeptide is an enzyme.  
     
     
         9 . A method of producing a substance which is the reaction product of an enzyme, the method comprising incorporating a DNA sequence coding for the enzyme into a gene of a mammal 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, whereafter it catalyses the formation of the reaction product from one or more substrates of the enzyme.  
     
     
         10 . A method as claimed in  claim 9 , wherein the reaction product is recovered from milk of the adult female mammal.  
     
     
         11 . A method as claimed in  claim 1  or  9  wherein the DNA sequence coding for the polypeptide is incorporated in vitro into a whey protein gene which is expressible in the mammary gland of an adult female mammal to form a fusion gene and the fusion gene is incorporated into the germline by injection into a fertilized egg or a cell of an embryo of the mammal, whereafter the injected fertilized egg or embryo is allowed to develop into an adult female mammal.  
     
     
         12 . A method as claimed in  claim 11 , wherein the injection is made into a nucleus of a two-cell embryo.  
     
     
         13 . A method as claimed in  claim 11 , wherein the linear molecules of DNA are injected into a pro-nucleus or a nucleus of the egg.  
     
     
         14 . A method as claimed in  claim 1  or  9 , wherein the mammal is a domestic livestock mammal.  
     
     
         15 . A method as claimed in  claim 14 , wherein the mammal is selected from members of the family Suidae, members of the genus Ovis, members of the genus Capra, and members of the genus Bos.  
     
     
         16 . A method as claimed in  claim 1  or  9 , wherein the mammal is a dairy sheep.  
     
     
         17 . A method as claimed in  claim 1  or  9 , wherein the whey protein gene encodes beta-lactoglobulin.  
     
     
         18 . A method as claimed in  claim 11 , wherein the fusion gene comprises a promoter.  
     
     
         19 . A method as claimed in  claim 11  or  18 , wherein the fusion gene comprises a start site for transcription.  
     
     
         20 . A method as claimed in  claim 11  or  18 , wherein the fusion gene comprises one or more distal 5′ regulatory sequences of a milk protein gene.  
     
     
         21 . A method as claimed in  claim 11  or  18 , wherein the fusion gene comprises a structural milk protein gene sequence optionally including internal regulatory sequences.  
     
     
         22 . A method as claimed in  claim 11  or  18 , wherein the fusion gene comprises a 3′ sequence flanking a milk protein gene.  
     
     
         23 . A method as claimed in  claim 11  or  18 , wherein the fusion gene comprises a cDNA sequence coding for the peptide of interest inserted into the first exon of a gene encoding a milk protein.  
     
     
         24 . A method as claimed in  claim 11  or  claim 18 , wherein the fusion gene includes some of the 5′ flanking sequences of a gene encoding a milk protein.  
     
     
         25 . A method as claimed in  claim 11  or  claim 18 , wherein the fusion gene contains a signal peptide for the peptide of interest.  
     
     
         26 . A method as claimed in  claim 1  or  9 , wherein a sequence encoding a signal peptide of the milk whey protein gene is precisely fused to that portion of the DNA sequence that encodes the N-terminal amino acid of the mature polypeptide in the substance produced.  
     
     
         27 . A method as claimed in any one of claims  1  or  9 , wherein the 3′ end of the DNA sequence terminates after its stop codon, but before its own polyA addition site.  
     
     
         28 . A method of producing a substance comprising a polypeptide substantially as herein described.  
     
     
         29 . A genetic construct comprising a DNA sequence encoding a polypeptide incorporated into a gene of a mammal tht codes for a milk whey protein in such a way that the DNA sequence is expressible in the mammary gland of the adult female mammal.  
     
     
         30 . An animal cell including a genetic construct as claimed in  claim 29 .  
     
     
         31 . An animal cell as claimed in  claim 30  which is an embryo cell.  
     
     
         32 . An animal whose genetic material includes a genetic construct as claimed in  claim 29 .  
     
     
         33 . A plasmid comprising a genetic construct as claimed in  claim 29.