Expression of human protein C in mammary tissue of transgenic mammals
First Claim
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1. A transgenic non-human mammal that contains and expresses a human protein C DNA construct in the cells of its mammary gland, wherein the DNA construct consists of:
- (a) a mammary gland promoter,(b) a nucleotide sequence that encodes a signal peptide, wherein said signal peptide is effective in directing the secretion of an associated polypeptide into the milk of said transgenic non-human mammal, and wherein said signal peptide-encoding nucleotide sequence is operatively associated with said mammary gland promoter, and(c) a nucleotide sequence encoding human protein C that is operatively associated with said signal peptide-encoding nucleotide sequence,wherein human protein C is secreted into the milk of said transgenic non-human mammal, and when purified, said protein C has a specific activity more than about 80% of the specific activity of human protein C isolated from human plasma, as determined by an assay of protein C serine protease activity or anticoagulant activity, andwherein said non-human mammal is selected from the group consisting of mouse, pig, sheep, goat and cattle.
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Abstract
Recombinant protein C characterized by a high percentage of active protein can be obtained in the milk of transgenic mammals that incorporate DNAs according to the present invention. Transgenic mammals of the present invention are produced by introducing into developing embryos DNA that encodes protein C, such that the DNA is stably incorporated in the DNA of germ line cells of the mature mammals and inherited in normal, mendelian fashion.
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Citations
20 Claims
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1. A transgenic non-human mammal that contains and expresses a human protein C DNA construct in the cells of its mammary gland, wherein the DNA construct consists of:
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(a) a mammary gland promoter, (b) a nucleotide sequence that encodes a signal peptide, wherein said signal peptide is effective in directing the secretion of an associated polypeptide into the milk of said transgenic non-human mammal, and wherein said signal peptide-encoding nucleotide sequence is operatively associated with said mammary gland promoter, and (c) a nucleotide sequence encoding human protein C that is operatively associated with said signal peptide-encoding nucleotide sequence, wherein human protein C is secreted into the milk of said transgenic non-human mammal, and when purified, said protein C has a specific activity more than about 80% of the specific activity of human protein C isolated from human plasma, as determined by an assay of protein C serine protease activity or anticoagulant activity, and wherein said non-human mammal is selected from the group consisting of mouse, pig, sheep, goat and cattle. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A process for the production of protein C, comprising the steps of:
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(a) providing a transgenic non-human mammal that contains and expresses a human protein C DNA construct in the cells of its mammary gland, wherein the DNA construct consists of; (i) a mammary gland promoter, (ii) a nucleotide sequence that encodes a signal peptide, wherein said signal peptide is effective in directing the secretion of an associated polypeptide into the milk of said transgenic non-human mammal, and wherein said signal peptide-encoding nucleotide sequence is operatively associated with said mammary gland promoter, and (iii) a nucleotide sequence encoding human protein C that is operatively associated with said signal peptide-encoding nucleotide sequence, wherein human protein C is secreted into the milk of said transgenic non-human mammal, and when purified, said protein C has a specific activity more than about 80% of the specific activity of human protein C isolated from human plasma, as determined by an assay of protein C serine protease activity or anticoagulant activity, and wherein said non-human mammal is selected from the group consisting of mouse, pig, sheep, goat and cattle, (b) producing milk from said transgenic non-human mammal, (c) collecting said milk, and (d) isolating said protein C from said milk. - View Dependent Claims (9, 10, 11, 12, 13, 14)
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15. A transgenic non-human mammal that contains and expresses a human protein C DNA construct in the cells of its mammary gland, wherein the DNA construct consists of:
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(a) a mammary gland promoter selected from the group consisting of a whey acidic protein promoter, a casein promoter, a lactalbumin promoter and a β
-lactoglobulin promoter,(b) a nucleotide sequence that encodes a signal peptide, wherein said signal peptide is effective in directing the secretion of an associated polypeptide into the milk of said transgenic non-human mammal, and wherein said signal peptide-encoding nucleotide sequence is operatively associated with said mammary gland promoter, and (c) a nucleotide sequence encoding human protein C that is operatively associated with said signal peptide-encoding nucleotide sequence, wherein human protein C is secreted into the milk of said transgenic non-human mammal, and when purified, said protein C has a specific activity more than about 80% of the specific activity of human protein C isolated from human plasma, as determined by an assay of protein C serine protease activity or anticoagulant activity, and wherein said non-human mammal is selected from the group consisting of mouse, pig, sheep, goat and cattle. - View Dependent Claims (16, 17)
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18. A process for the production of protein C, comprising the steps of:
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(a) providing a transgenic non-human mammal that contains and expresses a human protein C DNA construct in the cells of its mammary gland, wherein the DNA construct consists of; (i) a mammary gland promoter selected from the group consisting of a whey acidic protein promoter, a casein promoter, a lactalbumin promoter and a β
-lactoglobulin promoter,(ii) a nucleotide sequence that encodes a signal peptide, wherein said signal peptide is effective in directing the secretion of an associated polypeptide into the milk of said transgenic non-human mammal, and wherein said signal peptide-encoding nucleotide sequence is operatively associated with said mammary gland promoter, and (iii) a nucleotide sequence encoding human protein C that is operatively associated with said signal peptide-encoding nucleotide sequence, wherein human protein C is secreted into the milk of said transgenic non-human mammal, and when purified, said protein C has a specific activity more than about 80% of the specific activity of human protein C isolated from human plasma, as determined by an assay of protein C serine protease activity or anticoagulant activity, and wherein said non-human mammal is selected from the group consisting of mouse, pig, sheep, goat and cattle, (b) producing milk from said transgenic non-human mammal, (c) collecting said milk, and (d) isolating said protein C from said milk. - View Dependent Claims (19, 20)
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Specification