EP0879293A1 - Cassettes pour l'expression dans des plantes de proteines stables au stockage - Google Patents

Cassettes pour l'expression dans des plantes de proteines stables au stockage

Info

Publication number
EP0879293A1
EP0879293A1 EP97914150A EP97914150A EP0879293A1 EP 0879293 A1 EP0879293 A1 EP 0879293A1 EP 97914150 A EP97914150 A EP 97914150A EP 97914150 A EP97914150 A EP 97914150A EP 0879293 A1 EP0879293 A1 EP 0879293A1
Authority
EP
European Patent Office
Prior art keywords
gene
promoter
expression cassette
plants
cassette according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97914150A
Other languages
German (de)
English (en)
Inventor
Udo Conrad
Ulrike Fiedler
Julian Phillips
Olga Artsaenko
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut fuer Pflanzengenetik und Kulturpflanzenforschung
Original Assignee
Institut fuer Pflanzengenetik und Kulturpflanzenforschung
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE1996104588 external-priority patent/DE19604588A1/de
Priority claimed from DE1996120804 external-priority patent/DE19620804A1/de
Application filed by Institut fuer Pflanzengenetik und Kulturpflanzenforschung filed Critical Institut fuer Pflanzengenetik und Kulturpflanzenforschung
Publication of EP0879293A1 publication Critical patent/EP0879293A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8221—Transit peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to cassettes for the expression of storage-stable proteins in plants, in particular single-chain antibody fragments in transgenic tobacco or pea plants.
  • the aim of the invention was to put seed-specific expression in transgenic plants on a basis suitable for substance production. Another aim is to create a biological basis for the use of simple and manageable harvesting and processing technology, above all to ensure that the gene product formed in the plant in the period between the immediate harvest and the subsequent extraction and purification steps in quantity and desired activity remains stable at normal temperatures without cooling.
  • the expression cassettes according to the invention contain a promoter (preferably a constitutive promoter such as the CaMV 35S promoter or a seed-specific promoter), the LeB4 signal peptide, the gene to be expressed and an ER retention signal.
  • a promoter preferably a constitutive promoter such as the CaMV 35S promoter or a seed-specific promoter
  • the LeB4 signal peptide the gene to be expressed and an ER retention signal.
  • cassettes The structure of the cassettes is shown schematically in Figures 1 and 2 using the example of a single-chain antibody gene (scFv gene).
  • scFv gene a single-chain antibody gene
  • the expression cassette according to Fig. 1 is preferably used for the expression of genes of single-chain antibody fragments. It is also advantageous to use genes from recombinant antibody fragments as a translation fusion
  • REPLACEMENT BUIP (RULE 26) other functional proteins such as a second recombinant antibody, enzymes, toxins, chromophores and binding proteins.
  • SEKDEL specific ER retention signal
  • Other retention signals which occur naturally in plant and animal proteins located in the ER, can also be used to construct the cassette.
  • the seed-specific expression is approximately 1.9% of the total soluble protein, the single-chain antibody expression starting from day 16 of the seed development.
  • the use of the USP promoter to construct the expression cassette is particularly advantageous. It becomes active earlier during seed development, increasing the time available to enrich the expressed product. The expression rate is therefore higher than for cassettes with the LeB4 promoter.
  • the expression cassettes are transferred into bacterial strains by electroporation.
  • the resulting recombinant clones are used to transform dicotyledonous plants.
  • Plants that express gene products are selected and grown as genetically stable lines.
  • the gene products (including single-chain antibody fragments) are extracted after the harvest, if necessary after the plant tissue has dried. Tobacco and pea plants are particularly suitable as dicotyledonous plants.
  • the invention enables substances that are otherwise difficult to access, e.g. B. immunoglobulins to express at a high expression rate in plants and thereby to make them available for biotechnological use. Surprisingly, it has been found that single-chain antibody fragments during storage, for. B. in tobacco seeds remain stable for a long time (at least one year).
  • the hybridoma cell line NQ 10 / 12.5 is characterized in that the secreted, directed against the antigen phenyloxazolone monoclonal antibody having a high affinity (dissociation constant lxl0 - ⁇ M) and the specific sequences of the immunoglobulin genes are available (Berek et al., 1985). This monoclonal antibody was the starting point for the construction of the single-chain antibody fragment -scFv-ox. First, mRNA was isolated from the hybridoma cells and rewritten into cDNA.
  • variable immunoglobulin genes VH and VK served as a template for the amplification of the variable immunoglobulin genes VH and VK with the specific primers VH1 BACK and VH FOR-2 for the heavy chain and VK2 BACK and MJK5 FON X for the light chain (Clackson et al., 1991).
  • the isolated variable immunoglobulin genes were the starting point for the construction of a single-chain antibody fragment (scFv).
  • scFv single-chain antibody fragment
  • three components VH, VK and a linker fragment were combined in a PCR reaction mixture and the scFv-ox was amplified (Fig. 3).
  • the constructed scFv-ox gene had a size of 735 bp.
  • the variable domains were fused together in the order VH-L-VL.
  • the functional characterization (antigen binding activity) of the constructed scFv-ox gene was carried out after expression in a bacterial system.
  • the scFv-ox was synthesized as a soluble antibody fragment (Hoogenboom et al., 1991).
  • the activity and the specificity of the antibody fragment constructed was checked in ELISA tests (Fig. 4).
  • the scFv gene was cloned downstream of the LeB4 promoter.
  • the LeB4 promoter isolated from Vicia faba shows a strictly seed-specific expression of various foreign genes in tobacco (Bäumlein et al., 1987, 1991).
  • the scFv gene was fused with a signal peptide sequence which ensures entry into the endoplasmic reticulum and the ER retention signal SEKDEL, which ensures that it remains in the ER (Wandelt et al., 1992). (Fig. 5).
  • the constructed expression cassette (construct 11) was cloned into the binary vector pGSGLUCl (Saito et al., 1990) and transferred into the Agrobacterium strain EHA 101 by electroporation. Recombinant agrobacterial clones were used for the subsequent transformation of Nicotiana tabacum. 70-140 tobacco plants were regenerated per construct. After self-fertilization, both mature and seeds from various stages of development were harvested from the regenerated transgenic tobacco plants. The soluble proteins were obtained from these seeds after extraction in an aqueous buffer system. The analysis of the transgenic plants of series 11 shows that the fusion of the scFv gene with the DNA sequence of the ER retention signal SEKDEL achieved a maximum accumulation of 1.9% scFv proteins in the mature seed (Table 1 ).
  • Tab. 1 Summary representation of the seed-specific construct used, the number of tested and transgenic plants, their average scFv protein expression in mature seeds and the antigen binding activity of the antibody fragments. The expression levels were determined by Western blot analysis, the specific binding activity by means of direct ELISA.
  • the scFv gene was cloned downstream of the USP promoter.
  • the USP promoter isolated from Vicia faba shows a strictly seed-specific expression of various foreign genes in tobacco (Fiedler et al., 1993).
  • a stable accumulation of large amounts of antibody fragments was achieved by transporting the scFv protein into the endoplasmic reticulum.
  • the scFv gene was merged with a signal peptide sequence which fuses entry into the endoplasmic reticulum and the ER retention signal SEKDEL, which ensures that it remains in the ER (Wandelt et al., 1992) (Fig. 6).
  • the constructed expression cassette was cloned into the binary vector pGSGLUCl (Saito et al., 1990) and transferred by electroporation into the agrobacterial strain EHA 101. Recombinant agrobacterial clones were used for the subsequent transformation of Nicotiana tabacum. After self-fertilization, both mature and seeds from various stages of development were harvested from the regenerated transgenic tobacco plants. The soluble proteins were obtained from these seeds after extraction in an aqueous buffer system. Analysis of the transgenic plants shows that single-chain antibody fragments were synthesized from day 10 of seed development by the fusion of the scFv gene with the DNA sequence of the ER retention signal SEKDEL under the control of the USP promoter.
  • the scFv gene was first fused with a signal peptide sequence that entry into the endoplasmic reticulum and the ER retention signal KDEL, which ensures that it remains in the ER (Wandelt et al., Plant J. 2, 181-192 (1992)) (Fig. 1).
  • the constructed expression cassette (construct 9) was cloned into the binary vector pGSGLUC1 (Saito et al., Plant Cell Rep. 8, 718-721 (1990)) and transferred into the Agroba / cterium strain EHA 101 by electroporation. Recombinant agrobacterial clones were used for the subsequent transformation of Nicotiana tabacum. About 100 tobacco plants were regenerated. Leaf material from various stages of development was removed from the regenerated transgenic tobacco plants. The soluble proteins were obtained from this sheet material after extraction in an aqueous buffer system. Subsequent analyzes (Western blot
  • Table 2 Summary representation of the construct used, which mediates ubiquitous expression of the scFv gene, the number of tested and transgenic plants, their average scFv protein expression in the leaf and the antigen binding activity of the antibody fragments in the leaf extracts. The expression levels were determined by Western blot analysis and the antigen binding activity using a direct ELISA.
  • REPLACEMENT BLADE (RULE 26) provided and incubated for 1 to 4 h at room temperature without the addition of protease inhibitors. It was shown that there was no detectable degradation of the antibody fragments in the leaf extracts within the period tested.
  • the cause of the stability can be the compartment-specific occurrence of the antibody fragments, which means that after homogenization of the tissue these are not in the soluble supernatant together with the proteases.
  • Example 1 In addition to the stability in the buffer system used for the extraction, the possibility of storing the scFv protein also plays an important role, since the harvested material can often not be processed directly.
  • Example 1 it has already been described that, when expressed in the semen, it can be stored at RT for at least one year without a measurable loss of the accumulated antibody or its activity.
  • the storage of green tissue while maintaining the amount and activity of antibodies is not to be expected without further ado. Nevertheless, this was checked in a series of drying tests. For this purpose, fully grown leaves were harvested from 5 plants in the 9 series. Part of the
  • Leaf was immediately frozen (storage at -20 ° C) and another dried at RT and stored under the same conditions for one week. In parallel, leaves were dried at 50 ° C and stored for three weeks at room temperature. The result of these investigations is shown in Fig. 7. It was found that the antibody fragments of the leaves dried at room temperature are still present even after storage for one week. In a subsequent ELISA with extracts of the dried leaves, the antigen binding activity of the antibody fragment could also be demonstrated. No differences were found between the sheet material stored at -20 ° C. and the sheet material dried and stored at room temperature. In contrast, no scFv protein could be detected in leaves dried at 50 ° C in a Western blot (Fig. 7). Plant growth was not affected by the production of the recombinant proteins.
  • the starting point of the investigations was a single-chain antibody fragment against the phytohormone abscisic acid expressed in tobacco plants (Artsaenko et al., Plant J. 8, 745-750 (1995)).
  • the amount and activity of the synthesized scFv potein were determined in Western blot analyzes and ELISA tests.
  • the foreign gene was expressed under the control of the CaMV 35S promoter as a translation fusion with the LeB4 sinus peptide (N-terminal) and the ER retention signal KDEL (C-terminal).
  • a stable accumulation of high amounts of active antibody fragment was achieved by transporting the scFv protein into the endoplasmic reticulum. After harvesting the leaf material, pieces of a leaf were frozen at -20 ° C, lyophilized or dried at room temperature. The soluble proteins were obtained from the respective leaf materials by extraction in an aqueous buffer and the scFv protein was purified by affinity chromatography. Equal amounts (Fig. 8B) of purified scFv protein (frozen, lyophilized and dried) were used to determine the activity of the antibody fragment (Fig. ⁇ A). The same antigen binding activities were found. Legend for the pictures:
  • Fig. 1 Schematic representation of the cassette for the storage-stable expression of the scFv gene in leaves of transgenic tobacco plants
  • Fig. 2 Schematic representation of the cassette for seed-specific expression of the scFv gene
  • Fig. 3 Schematic representation of the construction of the scFv-ox (V gene for V (variable) region, L linker).
  • Fig. 4 Functional characterization of the scFv-ox 9 in a direct ELISA.
  • Fig. 5 Schematic representation of the cassette for seed-specific expression of the scFv-ox gene.
  • Fig. 6 Schematic representation of the cassette for seed-specific expression of the scFv-aABA.
  • Fig. 7 Studies on the stability of the scFv protein after drying the leaves of the transgenic plants 9/21 and 9/22 at RT and at 50 ° C. The scFv fragment was detected by Western blot analysis. 40 ⁇ g of total soluble protein was applied. Lane 1: control plant SNN, lane 2: 100 ng scFv protein, lane 3: 9/21 RT before drying, lane 4: 9/21 RT
  • track 5 9/21 50 ° C before drying
  • track 6 9/21 50 ° C after drying
  • track 7
  • Fig. 8 Evidence of the preservation of the antigen binding activity of the antibody fragment scFv-ABA in leaves after drying or lyophilization using ELISA tests.
  • Fig. 3A shows the antigen binding activity of the ⁇ cFv protein purified from fresh (1), lyophilized (2) and dried leaves.
  • Fig. 3B the respective amounts of scFv protein (about 100 ng) that were used for the ELISA analyzes are determined by means of Western blot analyzes. The sizes of the protein molecular weight standards are shown on the left.

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Botany (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne des cassettes pour l'expression de produits géniques, stables au stockage, dans des feuilles et, spécifiquement, dans des semences, en particulier de fragments d'anticorps à chaîne unique dans des feuilles et des semences de tabac et de pois transgéniques. L'invention couvre les domaines d'application tels que biotechnologie, médecine (diagnostic et thérapie), contrôle des denrées alimentaires et des végétaux, agriculture. La cassette d'expression selon l'invention comprend des promoteurs constitutifs ou spécifiques des semences, le peptide-signal LeB4, un gène à exprimer et un signal de rétention ER. On utilise de préférence une cassette d'expression renfermant comme promoteur constitutif, le promoteur CaMV 35S, comme gène, le gène pour un fragment d'anticorps à chaîne unique et, comme signal de rétention ER, la séquence aminoacide KDEL.
EP97914150A 1996-02-08 1997-02-07 Cassettes pour l'expression dans des plantes de proteines stables au stockage Withdrawn EP0879293A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE1996104588 DE19604588A1 (de) 1996-02-08 1996-02-08 Expressionskassette für die samenspezifische Expression
DE19604588 1996-02-08
DE19620804 1996-05-23
DE1996120804 DE19620804A1 (de) 1996-05-23 1996-05-23 Kassette zur Expression von lagerstabilen Genprodukten in Blättern
PCT/DE1997/000285 WO1997029200A1 (fr) 1996-02-08 1997-02-07 Cassettes pour l'expression dans des plantes de proteines stables au stockage

Publications (1)

Publication Number Publication Date
EP0879293A1 true EP0879293A1 (fr) 1998-11-25

Family

ID=26022734

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97914150A Withdrawn EP0879293A1 (fr) 1996-02-08 1997-02-07 Cassettes pour l'expression dans des plantes de proteines stables au stockage

Country Status (5)

Country Link
US (1) US6403371B1 (fr)
EP (1) EP0879293A1 (fr)
JP (1) JP2000504567A (fr)
CA (1) CA2246242A1 (fr)
WO (1) WO1997029200A1 (fr)

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EP1019517B2 (fr) 1997-09-30 2014-05-21 The Regents of The University of California Fabrication de proteines dans des graines de plantes
US7297478B1 (en) 2000-09-22 2007-11-20 Large Scale Biology Corporation Creation of variable length and sequence linker regions for dual-domain or multi-domain molecules
US20030044417A1 (en) * 1999-09-24 2003-03-06 Mccormick Alison A. Self antigen vaccines for treating B cell lymphomas and other cancers
US7084256B2 (en) * 1999-09-24 2006-08-01 Large Scale Biology Corporation Self antigen vaccines for treating B cell lymphomas and other cancers
US20030044420A1 (en) * 1999-09-24 2003-03-06 Mccormick Alison A. Self antigen vaccines for treating B cell lymphomas and other cancers
EP1130104A1 (fr) * 2000-02-16 2001-09-05 Stichting Dienst Landbouwkundig Onderzoek Réduction de la dégradation des produits végétales in planta
AU2001253107A1 (en) * 2000-04-03 2001-10-15 Monsanto Technology Llc Expression and purification of bioactive, authentic polypeptides from plants
DE10033750A1 (de) * 2000-07-12 2002-01-31 Mpb Cologne Gmbh Molecular Pla Pathogenresistenz in Organismen
EP2390256A1 (fr) * 2001-05-30 2011-11-30 Agrisoma, Inc. Chromosomes artificiels de plantes, leurs utilisations et leurs procédés de préparation
DE10155862A1 (de) * 2001-11-14 2003-05-28 Ipk Inst Fuer Pflanzengenetik Produktion von rekombinanten Antikörpern mittels Fusion mit Elastin-ähnlichen Peptiden
DE50311379D1 (de) * 2002-09-10 2009-05-14 Sungene Gmbh & Co Kgaa Expressionskassetten zur expression von nukleinsäuren in kohlenhydrat-speichernden sink-geweben von pflanzen
AU2003282667A1 (en) * 2002-10-03 2004-04-23 Large Scale Biology Corporation Multimeric protein engineering
EP2256189A1 (fr) 2003-03-28 2010-12-01 National Institute Of Agrobiological Sciences Procédé de production d'un organe de stockage de plante avec une production élevée de dérivé de GLP-1 recombinant.
US7951557B2 (en) 2003-04-27 2011-05-31 Protalix Ltd. Human lysosomal proteins from plant cell culture
DE602004015980D1 (de) * 2003-06-17 2008-10-02 Sembiosys Genetics Inc Verfahren zur insulinproduktion in pflanzen
WO2012142106A1 (fr) 2011-04-11 2012-10-18 Targeted Growth, Inc. Identification et utilisation de mutants de krp dans des plantes

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DE3920034C3 (de) * 1988-09-19 1999-09-23 Inst Pflanzengenetik & Kultur Verfahren zum Einführen von DNS - Sequenzen in das Genom von höheren Pflanzen
US5202422A (en) * 1989-10-27 1993-04-13 The Scripps Research Institute Compositions containing plant-produced glycopolypeptide multimers, multimeric proteins and method of their use
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Also Published As

Publication number Publication date
WO1997029200A1 (fr) 1997-08-14
US6403371B1 (en) 2002-06-11
CA2246242A1 (fr) 1997-08-14
JP2000504567A (ja) 2000-04-18

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