Plant bioreactors

a bioreactor and plant technology, applied in the field of plants as bioreactors, can solve the problems of unfavorable immune response, unfavorable steps involving purification or processing, and low protein production cost, and achieve the effect of enhancing the effect of an immune respons

Inactive Publication Date: 2006-05-18
AGRI & AGRI FOOD +2
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  • Claims
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Benefits of technology

[0052] The present invention also discloses a method of enhancing the effect of an immune response against...

Problems solved by technology

However, one limitation with clinical use of the oral tolerance approach is the cost of protein production, as oral tolerance requires the ingestion of relatively large amounts of protein antigens, often in excess of quantities that can be provided by conventional protein synthesis or fermentation systems.
Clearly steps involving purification or processing are undesirable if ease of oral administration is to be maximized as well as minimizing any associated costs for production.
Other plant bioreactor systems based on canola (Rooijen et al., 1995), potato (Manson et al., 1996), rice and cassaya (Ma & Hein, 1995) do not offer this advantage.
However, many non-food crop plants contain high levels of secondary plant products making plant tissue obtained from these plants unsuitable for direct oral administration.
However, these methods are directed to the alteration of nicotine levels so that the levels of other alkaloids that affect the flavours and aroma of tobacco are not modified in any manner.
These plants are not ideal for use as a non-food crop plant as describe herein, since the levels of only a select group of alkaloids have been reduced.
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Method used

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Examples

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example 1

AFP Gene Construction and Expression

[0162] To maximize potential AFP production at the translational level, the native 5′-untranslated region of the fish cDNA was replaced with the 5′-untranslated leader region of the tobacco mosaic virus (TMV) (Richards et al., 1977 & 1978; Sleat et al., 1988). The fish signal peptide sequence was also replaced with that of the tobacco pathogenesis-related protein 1b (PR-1b) (Cornelissen et al., 1986; Sijmons et al., 1990; Denecke et al., 1990). Oligonucleotide cassettes were designed: three oligonucleotides, #1091 (SEQ ID NO:1), #1092 (SEQ ID NO:4), and #1309 (SEQ ID NO:2), which would hybridize to generate the complete TMV leader sequence (TMV Cassette: FIG. 10A); and five oligonucleotides #4361-#4365 (SEQ ID NOS: 3, 5, 6, 8 and 9, respectively) which in concert with oligonucleotides #1091 and #1092 (SEQ ID NOS:1 and 4) would hybridize to generate a linked TMV leader and PR-1b signal peptide DNA sequence (TMV-PR Cassette: FIG. 10B). The 5′ end o...

example 2

IL-4 Expression and Determination of Biological Activity

[0177] Mouse IL-4 is a glycoprotein of 140 amino acid residues including a putative 20-amino acid signal peptide, which has a molecular weight of 19 to 21 kDa, depending on the degree of glycosylation (Proc. Natl. Acad. Sci. USA 83: 2061-2065, 1986). Mouse IL-4 cDNA was first amplified with PCR using the primers:

(SEQ ID NO:13)5′-AAT CTCGAGCATATC CAC GGATGCGAC-3′and(SEQ ID NO:14)5′-ATAGGTACCGTAATCCATTTGC ATGATGC-3′,

and the PCR product encoding the full protein was isolated and ligated to a signal peptide encoding sequence from peanut peroxidase and the KDEL sequence (SEQ ID NO:22). To facilitate subsequent purification of the recombinant product from plant cells, a DNA sequence encoding 6 consecutive histidines was also included in the reconstituted IL-4 gene (SEQ ID NO:21). The reconstituted IL-4 cDNA was then used to replace the β-glucuronidase (GUS) gene in plasmid pTRL2-GUS composed of a CaMV35S promoter with a double en...

example 3

Interleukin-10 (IL-10) Expression and Determination of Biological Activity

[0185] Full length mRNA encoding human IL-10 (hIL-10) is 1601 nucleotides long, with a short 5′UTR of 30 nucleotides (nt) and a long 3′UTR of 1037 nt. The hIL-10 coding sequence is 534 base pairs long and codes for a 178 amino acid preprotein with a 18 amino acid secretory signal (Vieira et al., 1991). The full length human IL-10 cDNA is isolated from polyA RNA by RT-PCR and fully sequenced to ensure fidelity. In order to maximize expression levels and transgene protein production, the IL-10 cDNA is examined for codon usage patterns and optimized as necessary (Perlak et al., 1991).

[0186] Since ER retention signals can increase the concentration of disulfide bonded transgene proteins, therefore the KDEL ER retention motif may be added to the IL-10 cDNA using site directed mutagenesis (see results in FIG. 5C). For comparative purposes an identical synthetic gene, without the KDEL signal, is prepared and used i...

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Abstract

A plant expressing a cytokine and an autoantigen is disclosed. An example of a cytokine is a contra-inflammatory cytokine, for example IL-4 and IL-10, and an example of autoantigen is GAD. A novel method for the production of transgenic proteins of interest suitable for oral administration is also disclosed. Further, non-food crop plants expressing one or more proteins of interest are disclosed, as is a method involving the preparation of a protein of interest suitable for oral administration within a non-food crop plant comprising, transforming the non-food crop plant with a suitable vector containing a gene of interest and appropriate regulatory regions to ensure expression of the gene of interest within the non-food crop plant, such that the non-food crop plant is characterized as being non-toxic, non-addictive, palatable, and requiring minimal or no processing prior to oral administration. An example of a non-food crop plant is low alkaloid tobacco. Proteins of interest may include pharmaceutically active proteins such as growth regulators, insulin, interferons, interleukins, growth hormone, erythropoietin, G-CSF, GM-CSF, hPG-CSF, M-CSF, Factor VIII, Factor IX, tPA, antibodies, antigens and combinations and/or derivatives thereof.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This is a Division of application Ser. No. 10 / 137,647, filed May 3, 2002, pending; which is a Continuation In-Part of application Ser. No. 09 / 773,385, filed Feb. 1, 2001, abandoned; which is a Continuation of application Ser. No. 09 / 102,050, filed Jun. 22, 1998, abandoned; which is a Continuation In-Part of application Ser. No. 08 / 733,791, filed Oct. 18, 1996, abandoned; the entire contents of which are incorporated by reference herein.FIELD OF THE INVENTION [0002] The present invention relates to the use of plants as a bioreactor for the production of heterologous proteins. More specifically this invention relates to the expression of transgenes of interest for oral administration using plants. BACKGROUND OF THE INVENTION [0003] Expression of mammalian genes in several plants including tobacco and Arabidopsis has been recognized as efficient, low cost, non-sterile bioreactors for the production of proteins valuable to both medicine and...

Claims

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Application Information

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IPC IPC(8): A01H1/00A61K38/20A61K36/81C07K14/54C12N9/88C12N15/12C12N15/82
CPCA61K38/2026A61K39/0008C07K14/5406C07K14/5428C12N9/88C12N15/8242C12N15/8243C12N15/8257C12N15/8258C12N15/8273A61K2300/00
Inventor BRANDLE JIMMA SHENGWUMENASSA RIMAJEVNIKAR ANTHONYDELOVITCH TERRY
Owner AGRI & AGRI FOOD
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