Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal to manufacture... – The protein is isolated or extracted from milk
Reexamination Certificate
1998-02-24
2003-03-04
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal to manufacture...
The protein is isolated or extracted from milk
C800S014000, C435S069100, C536S023100, C536S023500
Reexamination Certificate
active
06528699
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the production and secretion of proteins which are not ordinarily secreted.
BACKGROUND OF THE INVENTION
A growing number of recombinant proteins are being developed for therapeutic and diagnostic applications. However, many of these proteins may be difficult or expensive to produce in a functional form and/or in the required quantities using conventional methods. Conventional methods involve inserting the gene responsible for the production of a particular protein into host cells such as bacteria, yeast, or mammalian cells, e.g., COS cells, and then growing the cells in culture media. The cultured cells then synthesize the desired protein. Traditional bacteria or yeast systems may be unable to produce many complex proteins in a functional form. While mammalian cells can reproduce complex proteins, they are generally difficult and expensive to grow, and often produce only mg/L quantities of protein. In addition, non-secreted proteins are relatively difficult to purify from procaryotic or mammalian cells as they are not secreted into the culture medium.
SUMMARY OF THE INVENTION
In general, the invention features, a method of making and secreting a protein which is not normally secreted (a non-secreted protein). The method includes expressing the protein from a nucleic acid construct which includes:
(a) a promoter, e.g., a mammary epithelial specific promoter, e.g., a milk protein promoter;
(b) a signal sequence which can direct the secretion of a protein, e.g. a signal sequence from a milk specific protein;
(c) optionally, a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein, e.g., a protein secreted into milk, to allow secretion, e.g., in the milk of a transgenic mammal, of the non-secreted protein; and
(d) a sequence which encodes a non-secreted protein, wherein elements (a), (b), optionally (c), and (d) are preferably operatively linked in the order recited.
In preferred embodiments: elements a, b, c (if present), and d are from the same gene; the elements a, b, c (if present), and d are from two or more genes.
In preferred embodiments the secretion is into the milk of a transgenic mammal.
In preferred embodiments: the signal sequence is the &bgr;-casein signal sequence; the promoter is the &bgr;-casein promoter sequence.
In preferred embodiments the non-secreted protein-coding sequence: is of human origin; codes for a truncated, nuclear, or a cytoplasmic polypeptide; codes for glutamic acid decarboxylase or myelin basic protein.
In preferred embodiments, the protein is a mutant protein which lacks a biological activity of the wild type protein.
In another aspect, the invention features, a nucleic acid construct, preferably an isolated nucleic acid construct, which includes:
(a) a promoter, e.g., a mammary epithelial specific promoter, e.g., a milk protein promoter;
(b) a signal sequence which can direct the secretion of a protein, e.g., a signal sequence from a milk specific protein;
(c) optionally, a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein, e.g., a protein secreted into milk, to allow secretion, e.g., in the milk of a transgenic mammal, of the non-secreted protein; and
(d) a sequence which encodes a non-secreted protein, wherein elements (a), (b), optionally (c), and (d) are preferably coupled in the order recited.
In preferred embodiments: elements a, b, c (if present), and d are from the same gene; the elements a, b, c (if present), and d are from two or more genes.
In preferred embodiments the secretion is into the milk of a transgenic mammal.
In preferred embodiments: the signal sequence is the &bgr;-casein signal sequence; the promoter is the &bgr;-casein promoter sequence.
In preferred embodiments the non-secreted protein-coding sequence: is of human origin; codes for a truncated, nuclear, or a cytoplasmic polypeptide; codes for glutamic acid decarboxylase or myelin basic protein.
In preferred embodiments, the protein is inactive, e.g., it is a mutant protein which lacks a biological activity of the wild type protein.
In another aspect, the invention features, a method for providing a non-secreted protein, e.g., a heterologous non-secreted polypeptide, in the milk, of a transgenic mammal. The method includes obtaining milk from a transgenic mammal having introduced into its germline a nucleic acid construct described herein, e.g., a heterologous non-secreted protein-coding sequence operatively linked to a signal and a promoter sequence that result in the preferential expression of the protein-coding sequence in mammary gland epithelial cells, thereby secreting the heterologous non-secreted polypeptide in the milk of the mammal.
In preferred embodiments, the protein is inactive, e.g., it is a mutant protein which lacks a biological activity of the wild type protein.
In preferred embodiments the transgenic mammal is selected from the group consisting of sheep, mice, pigs, cows and goats. The preferred transgenic mammal is a goat.
In preferred embodiments, the promoter is selected from the group consisting of the beta lactoglobulin promoter, whey acid protein promoter, &bgr;-casein promoter and the lactalbumin promoter. The preferred promoter is the &bgr;-casein promoter.
In preferred embodiments, the signal sequence is &bgr;-casein signal sequence.
In preferred embodiments, the non-secreted protein-coding sequence: is of human origin; codes for a truncated, nuclear, or a cytoplasmic polypeptide; codes for glutamic acid decarboxylase or myelin basic protein; codes for an inactive form of glutamic acid decarboxylase.
In preferred embodiments, the protein is fused to other sequences, e.g., to one or both of: a signal sequence, e.g., the signal sequence of &bgr;-casein and/or a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein, e.g., a protein secreted into milk, to allow secretion, e.g., in the milk of a transgenic mammal, of the non-secreted protein.
In other preferred embodiments the non-secreted polypeptide is purified from the milk of a transgenic mammal.
In another aspect, the invention features, a method of inducing tolerance in a subject to an antigen. The antigen can be a xenoantigen, an alloantigen or a autoantigen. The antigen can be a protein, e.g., a non-secreted protein.
The method includes:
providing a tolerogen expressed in a transgenic mammal which includes the antigen;
and administering the tolerogen to the subject in an amount sufficient to induce tolerance to said protein antigen.
In preferred embodiments: the tolerogen is administered, preferably orally, to the subject in the milk of a transgenic mammal, e.g., a transgenic dairy mammal, e.g., a goat, sheep, or cow. Other mammals, e.g., pigs, can also be used.
In preferred embodiments, the tolerogen is or includes: a protein, e.g., an inactive protein; a non-secreted protein; a fusion of a non-secreted protein and another peptide sequence, e.g., a protein antigen fused to all or part of a secreted protein.
Preferably, the transgenically produced product, is in inactive form, e.g., it is a mutant which lacks an activity of the wild type protein.
In preferred embodiments, the autoantigen is fused to other sequences, e.g., to one or both of: a signal sequence, e.g., the signal sequence of &bgr;-casein and/or a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein, e.g. a protein secreted into milk, to allow secretion, e.g., in the milk of a transgenic mammal, of the non-secreted protein.
In preferred embodiments, the antigen is: an antigen which is characteristic of an autoimmune disorder, e.g., diabetes, lupus, multiple sclerosis, rheumatoid arthritis; GAD; MBP; a transcription factor.
In preferred embodiments the subject is at risk of developing, or has, an autoimmune disorder, e.g., diabetes, lupus, multiple sclerosis, of rheumatoid arthritis.
In yet another aspect, the invention features, a method of treating insulin-dependent diabetes mellittis
Chen Li-How
DiTullio Paul
Meade Harry
Crouch Deborah
Fish & Richardson P.C.
Genzyme Transgenics Corporation
Ton Thaian
LandOfFree
Transgenically produced non-secreted proteins does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Transgenically produced non-secreted proteins, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Transgenically produced non-secreted proteins will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3056912