Food or edible material: processes – compositions – and products – Products per se – or processes of preparing or treating... – Basic ingredient lacteal derived other than butter...
Reexamination Certificate
1998-06-15
2001-04-03
Hauda, Karen M. (Department: 1632)
Food or edible material: processes, compositions, and products
Products per se, or processes of preparing or treating...
Basic ingredient lacteal derived other than butter...
C530S399000, C530S397000
Reexamination Certificate
active
06210736
ABSTRACT:
The invention relates to transgenic prolactin, and methods of making and using transgenic prolactin.
BACKGROUND OF THE INVENTION
A growing number of recombinant proteins are being developed for therapeutic, diagnostic, agricultural, veterinary, nutritional and other applications; however, many of these proteins may be difficult or expensive to produce in a functional form in the substantial quantities using conventional methods.
Conventional methods often involve inserting the gene responsible for the production of a particular protein into host cells such as bacteria, yeast, or mammalian cells. The cells are grown in culture medium and the desired protein is recovered from the cells or the culture medium. Traditional bacteria or yeast systems are sometimes unable to produce a complex protein in finctional form. While some mammalian cells can reproduce complex proteins, they are often difficult and expensive to grow, and produce only protein in relatively low amounts. In addition, non-secreted proteins are relatively difficult to purify from procaryotic or mammalian cells, as they are often not secreted into the culture medium.
SUMMARY OF THE INVENTION
In general, the invention features, a transgenically produced preparation of prolactin, preferably human prolactin.
The transgenically produced prolactin is produced in a transgenic organism, i.e., a transgenic plant or animal. Preferred tralsgenic animals include: mammals; birds; reptiles; and amphibians. Suitable mammals include: ruminants; ungulates; domesticated mammals; and dairy animals. Particularly preferred animals include: goats, sheep, camels, cows, pigs, horses, oxen, and llamas. Suitable birds include chickens, geese, and turkeys. Where the transgenic protein is secreted into the milk of a transgenic animal, the animal should be able to produce at least 1, and more preferably at least 10, or 100, liters of milk per year.
In preferred embodiments, less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 2.5%, or 1% of the prolactin molecules in a transgenically made preparation, preferably as it is made in the transgenic organism, are glycosylated.
In preferred embodiments, the transgenically produced prolactin preparation, preferably as it is made in the transgenic organism, is less than 50%, 40%, 30%, 20%, 10%, or 5%, glycosylated (in terms of the number of molecules in a preparation which are glycosylated, or in terms of the total contribution of sugar to the molecular weight in a preparation) AS compared to the glycosylation of prolactin as it is found or as it is isolated from naturally occurring nontransgenic source, or as it is isolated from recombinantly produced prolactin in cell culture.
In preferred embodiments the transgenic preparation, preferably as it is made in the transgenic organism, includes glycosylated and non-glycosylated forms, and some or all of the glycosylated forms are removed, e.g., from a body fluid, e.g., milk, e.g., by standard protein separation methods.
In preferred embodiments, the transgenically produced prolactin is made in a mammary gland of the transgenic mammal, e.g., a ruminant, e.g., a goat.
In preferred embodiments, the transgenically produced prolactin is secreted into the milk of the transgenic mammal, e.g., a ruminant, e.g., a goat.
In preferred embodiments, the transgenically produced prolactin is made under the control of a mammary gland specific promoter, e.g., a milk specific promoter, e.g., a milk serum protein or casein promoter. The milk specific promoter can is a casein promoter, beta lactoglobulin promoter, whey acid protein promoter, or lactalbumin promoter.
In preferred embodiments, the prolactin is made under the control of a bladder, or egg specific promoter and prolactin is secreted into the urine or into an egg.
In preferred embodiments, the transgenically produced prolactin preparation differs in average molecular weight, activity, clearance time, or resistance to proteolytic degradation from non-transgenic forms.
In preferred embodiments, the glycosylation of the transgenically produced prolactin preparation differs from prolactin as it is found or as it is isolated from recombinantly produced prolactin in cell culture, or transgenic prolactin produced in mouse.
In preferred embodiments, the transgenically produced prolactin is expressed from a transgenic organism and the glycosylation of the transgenically produced prolactin preparation differs from the glycosylation of prolactin as it is found or as it is isolated from a bacterial cell, a yeast cell, an insect cell, a cultured mammalian cell, e.g., a CHO, COS, or HeLa cell. For example, it is different from a protein made by a cultured mammalian cell which has inserted into it a nucleic acid which encodes or directs the expression of prolactin.
In preferred embodiments, the prolactin is expressed from a transgenic mammal other than a rodent, e.g., mouse. For example, the prolactin is expressed from a goat, and the glycosylation of the transgenically produced prolactin differs from the glycosylation of prolactin as it is found or as it is isolated from a transgenic rodent which has inserted into it a nucleic acid which encodes or directs the expression of prolactin.
In preferred embodiments, the electrophoretic mobility of the prolactin preparation, e.g., as determined by SDS-PAGE, is different from the electrophoretic mobility of a naturally occurring human prolactin; the electrophoretic mobility of the preparation is different from the electrophoretic mobility of a recombinantly produced human prolactin produced in mammalian cells, e.g., CHO, COS, or HeLa cells, or procaryotic cells, e.g., bacteria, or yeast, or insect cells.
In preferred embodiments, the prolactin differs by at least one amino acid residue from a naturally occurring human prolactin; the prolactin differs by at least one amino acid residue from a recombinantly produced human prolactin produced in mammalian cells, e.g., CHO, COS, or HeLa cells, procaryotic cells, e.g., bacteria, or yeast, or insect cells.
In preferred embodiments, the prolactin the amino acid sequence is that of mammalian or primate, preferably human, prolactin.
In preferred embodiments, the preparation includes at least 1, 10, or 100 milligrams of prolactin. In preferred embodiments, the preparation includes at least 1, 10, or 100 grams of prolactin.
In preferred embodiments, the preparation includes at least 1, 10, 100, or 500 milligrams per milliliter of prolactin.
In another aspect, the invention features, an isolated nucleic acid molecule including a prolactin protein-coding sequence operatively linked to a tissue specific promoter, e.g., a mammary gland specific promoter sequence that results in the secretion of the protein in the milk of a transgenic mammal.
In preferred embodiments, the promoter is a milk specific promoter, e.g., a milk serum protein or casein promoter. The milk specific promoter can is a casein promoter, beta lactoglobulin promoter, whey acid protein promoter, or lactalbumin promoter.
In preferred embodiments, the promoter is a bladder, or egg specific promoter and prolactin is secreted into the urine or into an egg.
In preferred embodiments, the prolactin the amino acid sequence is that of mammalian or primate, preferably human, prolactin.
In another aspect, the invention features, a method of making transgenic prolactin or a preparation of transgenic prolactin. The method includes:
providing a transgenic organism, i.e., a transgenic animal or plant, which includes a transgene which directs the expression of prolactin, preferably human prolactin;
allowing the transgene to be expressed; and recovering transgenically produced prolactin or a preparation of transgenically produced prolactin, from the organism or from a product produced by the organism, e.g., milk, seeds, hair, blood, eggs, or urine.
In preferred embodiments, the method further includes:
inserting a nucleic acid which directs the expression of prolactin into a cell and allowing the cell to give rise to a transgenic organism;
Preferred transgenic animals include: mammals; birds; reptiles; a
Echelard Yann
Wilburn Brian
Fish & Richardson P.C.
Genzyme Transgenics Corporation
Hauda Karen M.
Shukla Ram R.
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