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Expression of proteins in milk

a technology of recombinant proteins and milk, which is applied in the direction of viruses/bacteriophages, peptide sources, etc., can solve the problems of high cost, unreliability, and high cost of recombinant production in prokaryotic cells, and achieves low cost and high level production

Inactive Publication Date: 2002-09-12
PHARMING INTPROP BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0004] The present invention solves such problems by providing an efficient means of producing large quantities of recombinant protein products in the milk of transgenically altered mammals. According to this invention, a DNA sequence coding for a desired protein is operatively linked in an expression system to a milk-specific protein promoter, or any promoter sequence specifically activated in mammary tissue, through a DNA sequence coding for a signal peptide that permits secretion and maturation of the desired protein in the mammary tissue. More preferably, the expression system also includes a 3' untranslated region downstream of the DNA sequence coding for the desired recombinant protein. This untranslated region may stabilize the rDNA transcript of the expression system. Optionally, the expression system also includes a 5' untranslated region upstream of the DNA sequence coding for the signal peptide.
[0005] The expression system is transgenically introduced into a host genome by standard transgenic techniques. As a result, one or more copies of the construct or system becomes incorporated into the genome of the transgenic mammal. The presence of the expression system will permit the female species of the mammal to produce and to secrete the recombinant protein product, into or along with its milk. Such method permits the low cost, high level production of the desired proteins.

Problems solved by technology

As a result, the recombinant production of such products in prokaryotic cells has proven to be less than satisfactory because the desired recombinant proteins are incorrectly processed, lack proper glycosylation or are improperly folded.
This technique has proven to be both expensive and often unreliable due the variability of cell culture methods.
For example, average yields are 10 mg of recombinant protein per liter of culture media, with the resulting cost typically for exceeding $1,000 per gram of recombinant protein.

Method used

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  • Expression of proteins in milk

Examples

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Effect test

example 1

[0022] Bovine Alpha S-1 Casein

[0023] We cloned bovine alpha S-1 casein with a cosmid library of calf thymus DNA in the cosmid vector HC79 (from Boehringer Mannheim) as described by B. Hohn and J. Collins, Gene, 11, pp. 291-98 (1980). The thymus was obtained from a slaughterhouse and the DNA isolated by standard techniques well known in the art (T. Maniatis et al., "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory at page 271 (1982)). We isolated the cosmid library using standard techniques (F. Grosveld et al., Gene, 13, pp. 227-31 (1981)). We partially digested the calf thymus DNA with Sau3A (New England Bio Labs) and ran it on a salt gradient to enrich for 30 to 40 kb fragments. The partially digested DNA fragments were then ligated with BamHI digested HC79 cosmid vector, followed by in vitro packaging by lambda extracts (Amersham) following the manufacturer's recommendation. The in vitro packaged material was then used to infect the E.coli K-12 strain HB101 f...

example 2

Construction of the Cas-recombinant Product Construct

[0030] One recombinant protein that can be produced by the process of this invention is tissue plasminogen activator or TPA. As demonstrated below, the casein signal peptide was used to direct secretion of TPA from the mammary glands of transgenic mice carrying a construct according to this invention. In this construct, the nucleotide sequence of the casein signal peptide was fused to the sequence of mature TPA by RNA processing. The sequence of TPA has been described in D. Pennica et al., Nature, 301, pp. 214-21 (1983). In the TPA gene, as in the CAS gene, there is a BamHI site in Intron II which separates the signal peptide from the mature sequence [R. Fisher et al., J. Biol. Chem., 260, pp. 11223-30 (1985)]. The cDNA of TPA shows the BglII site in Exon III at amino acid #3 of mature TPA.

[0031] We subcloned a 1.7 kb fragment from tha genomic clone of TPA [R. Fisher et al., supra] using BamHI-BglII. The 1.7 kb fragment contained ...

example 3

Transgenic Incorporation of the Construct into Mice

[0033] The procedure for transgenic incorporation of the desired genetic information into the developing mouse embryo is established in the art [B. Hogan et al., "Manipulating The Mouse Embryo: A Laboratory Manual" Cold Spring Harbor Laboratory (1986)]. We used an F1 generation (Sloan Kettering) cross between C57B1 and CB6 (Jackson Laboratories). Six week old females were superovulated by injection of Gestile (pregnant mare serum) followed by human chorionic gonadotropin two days later. The treated females were bred with C57B1 stud males 24 hours later. The preimplantation fertilized embryos were removed within 12 hours following mating for microinjection with DNA and implantation into pseudopregnant females.

[0034] We injected the construct by first digesting the cumulus cells surrounding the egg with Hyaluronidase. The construct was injected into the pronucleus of the embryo until it swelled 30% to 50% in size. We then implanted th...

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Abstract

This invention relates to the production of recombinant proteins in mammals' milk. Particularly, this invention relates to an expression system which when transgenically incorporated into a mammal permits the female species of that mammal to produce the desired recombinant protein in or along with its milk. This invention also relates to the transgenic mammal that produces the desired recombinant product in its milk.

Description

TECHNICAL FIELD OF INVENTION[0001] This invention relates to the production of recombinant proteins in mammals' milk. Particularly, this invention relates to an expression system which comprises at least a milk-specific protein promoter operatively linked to a DNA sequence coding for a signal peptide and a desired recombinant protein product. When such a system is transgenically incorporated into a mammal, the recombinant protein is expressed in the milk of the lactating transgenic mammal. This invention also relates to the transgenic mammal that produces the desired recombinant product in its milk. Recombinant products produced by the expression systems and transgenically altered mammals of this invention can be produced at significantly less cost than by conventional recombinant protein production techniques.BACKGROUND ART[0002] Recombinant DNA technology has enabled the cloning and expression of genes encoding medically and agriculturally important proteins and glycoproteins. Suc...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/435A01K67/027C07K14/47C07K14/745C07K14/755C07K14/76C12N9/72C12N15/09C12N15/85C12P21/00
CPCA01K67/0275A01K67/0278A01K2207/15A01K2217/00A01K2217/05A01K2227/103A01K2227/105A01K2267/01C07K14/4732C07K2319/00C07K2319/02C12N9/6456C12N15/85C12N15/8509C12N2830/008C12N2830/85C12N2840/44Y10S530/832Y10S530/833
Inventor MEADE, HARRYLONBERG, NILS
Owner PHARMING INTPROP BV
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