WO2001020974A1 - Expression quantitative de proteines transitoire dans une culture de tissu vegetal - Google Patents
Expression quantitative de proteines transitoire dans une culture de tissu vegetal Download PDFInfo
- Publication number
- WO2001020974A1 WO2001020974A1 PCT/US2000/026011 US0026011W WO0120974A1 WO 2001020974 A1 WO2001020974 A1 WO 2001020974A1 US 0026011 W US0026011 W US 0026011W WO 0120974 A1 WO0120974 A1 WO 0120974A1
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- WO
- WIPO (PCT)
- Prior art keywords
- plant
- culture
- agrobacterium
- polypeptide
- plant tissue
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- 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)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- 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)
- C12N15/8241—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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/13—Immunoglobulins specific features characterized by their source of isolation or production isolated from plants
Definitions
- the present invention also relates to the production of large-scale amounts of the
- polypeptide by mixing together plant tissue and recombinant microorganisms under
- polypeptides such as monoclonal antibodies (MAbs), human growth hormone (hGH), blood transport proteins, including
- the present application is directed to plant cell and root culture as a platform for
- Pilot-scale production of plant biomass would be a
- transgenic plants as a means of producing biochemicals.
- the process involves the growth of plant suspended cells or root tissue within a bioreactor. This tissue acts as the expression host for transient expression
- insect cell culture systems The process involves the addition of a live bacterial or viral
- tissue may be used.
- the present invention has met the hereinbefore described need.
- the present application describes technology related to utilizing cultured plant tissues
- An aspect of the present invention is on transient protein
- bioreactor systems provides the capability of introducing the DNA to tissue that is of a
- the plant tissue can be genetically altered to provide unique protein processing
- the invention has particular utility in the area of discovery and development of
- transgenic plant development establishing transgenic plants, which express heterologous
- the method of the present application reduces the time
- mammalian culture cell baculovirus and so on
- transiently producing a polypeptide in a plant tissue comprising:
- nucleotide sequence is transferred to the plant
- the process is directed to large-scale transient expression
- the plant tissue sample may include a plant cell, algal cell
- the plant may be a dicot or a monocot. In the case of a
- the plant may include, but is not limited to, tobacco, potato, bean and soybean. In the
- the plant may include, but is not limited to, corn.
- the Agrobacterium may be any suitable organism. Further, according to the method of the invention, the Agrobacterium may be any suitable organism.
- Agrobacterium tumefaciens or Agrobacterium rhizogenes Preferably, the Agrobacterium
- auxotroph that is deficient in its ability to metabolize amino acids, vitamins, and/or
- nucleic acid precursors If it is deficient for example in amino acid metabolism, it is desirable.
- amino acid be a low cost amino acid, and not present in the co-culture medium in
- the polypeptide of interest may be a protein such as an
- enzyme enzyme, antibody or a therapeutic protein. It may also be a biomaterial or a gene product
- transcriptional factor transcriptional factor, signal molecule, receptor, etc.
- the method of the invention further includes monitoring and controlling the
- bioreactor environment Some of the factors to be monitored and controlled include, but are
- the pH may be
- the Agrobacterium may be an auxotroph and be added to
- the Agrobacterium can be an auxotroph and can be added to plant culture at a biomass
- the time length of reaction between the plant culture and the Agrobacterium may be about 1
- transient expression conditions determined in volumes of about 50 ml to about 1 liter
- activator may be added to the mixture of plant culture and Agrobacterium culture.
- activator may be acetosyringone, syringaldehyde or other phenolic compounds that interact
- FIG. 1 shows growth of Agrobacterium rhizogenes transformed root cultures of
- Nicotiana benthemiana (N.b.) after exposure to tobacco mosaic virus. Growth rates assessed as
- Gamborg B5 medium extension measured daily by tracing roots through the bottom of the Petri
- FIG. 2 shows a growth time course plot for root cultures of Hyoscyamus muticus grown
- Roots were inoculated in 50 mL of B5
- FIG. 3 shows the level of plant-expressed GUS ( ⁇ -glucuronidase) at different times
- the Agrobacterium EHA105::GUS-intron suspension reached an OD of approximately 0.75
- the initial bacterial titer in the NGA-bacteria mixture was 1% v/v.
- roots were harvested, frozen with liquid nitrogen, ground with a mortar and pestle and
- glucuronidase a quantitative approach.
- GUS Protocols using GUS gene as a reporter of
- methylumbelliferyl- ⁇ -D-glucuronide (MUG) was added to the cell extract aliquot and
- MU methylcoumarin
- FIG. 4 shows a graph of the results of an Agrobacterium auxotroph screen for growth
- chromosomal background auxotrophs The naming convention used is to designate the
- auxotrophs by their required supplement, where aux refers to an unknown requirement.
- Agrobacterium tumefaciens EHA105::GUS-intron was used as a control.
- titer in the NGA-bacteria mixture was approximately 10% v/v.
- FIG. 5 shows a graph of the level of plant-expressed GUS in roots wounded for
- Nicotiana glutinosa (NGA )roots were wounded in a lab blender
- Agrobacterium tumefaciens EHA105::GUS-intron were grown in a baffled flask and
- EHA105::GUS-intron suspension reached an OD of approximately 0.75 at the time of
- the initial bacterial titer in the NGA-bacteria mixture was 1% v/v based on a
- the media was changed after approximately 14 hours of co-culture to
- FIG. 8 for GUS enzyme. For a nominal cell concentration of 300 g FW of cells/L this would be a nominal cell concentration of 300 g FW of cells/L this would be a nominal cell concentration of 300 g FW of cells/L this would be a nominal cell concentration of 300 g FW of cells/L this would be a nominal cell concentration of 300 g FW of cells/L this would be a nominal cell concentration of 300 g FW of cells/L this would be a nominal cell concentration of 300 g FW of cells/L this would be used.
- FIG. 6 shows a graph of the level of plant-expressed GUS in cells wounded for
- EHA105::GUS-intron suspension reached an OD of 1 at the time of infection.
- FIG. 7 shows the level of plant-expressed GUS in hairy and hairless roots. Twelve-
- T or T/PBA-bacteria mixture was 1% v/v based on a bacterial OD equal to 1.
- acetosyringone was added to the co-culture media as a bacterial activator.
- the media was
- FIG. 8 shows the effect of pH of the surrounding medium on the expression of GUS
- Tobacco BY2 cells were 5 days old. Cells were filtered and re-
- FIG. 9 shows the effect of pH together with activation by acetosyringone on the level
- bacterial titer in the NGA-bacteria mixture was 1 % v/v based on a bacterial OD equal to 1.
- the media was changed after approximately 17 hours of co-culture to minimize bacterial
- FIG. 10 shows the effect of pH together with activation with syringaldehyde on the
- fresh media adjusted to a pH of about 4.9, 5.2, 5.5, 5.8, or 6.1, containing 100 microM
- the Agrobacterium suspension reached an OD of approximately 1 at the
- the initial bacterial titer in the NGA-bacteria mixture was 1 % v/v based on
- the invention is directed to achieving transient protein expression in plant tissue
- transient means the expression of a recombinant
- transgenic cells where heterologous DNA is introduced into the plant chromosome.
- polypeptide of interest typically, about 10 L to over about 1000 L volume of cells is used,
- polypeptide is isolated and purified. More preferably, from a
- volume of about 100 L, about 100 mg amount of polypeptide is isolated. Even more
- a “bioreactor” may be any container in which a reaction between the
- plant and Agrobacterium may occur to produce transiently expressed polypeptides.
- the container is about 50 ml to 10,000 liters in volume. More preferably, the volume is about 1 liter to 10,000 liters. Even more preferably, the volume is about 10 liters to
- a “bioreactor” may be any container in which a reaction between the
- plant and Agrobacterium may occur to produce transiently expressed polypeptides.
- the container is about 50 ml to about 10,000 liters in volume. More preferably, the
- volume is about 1 liter to about 10,000 liters. Even more preferably, the volume is about 100
- DW / L or plant tissue Preferably, about 20 g DW / L or plant tissue are used. Even more preferably, about 40 g
- DW / L or plant tissue are used as determined by the bioreactor monitoring and control
- the amount of the bacteria is about 5 x 10 4 cells / mL.
- the amount of bacteria is about 1 x 10 4 cells / mL.
- the pH of the environment influences the transfer of DNA to plants for transient
- an effective pH is from about 4.9 to about 6.1 depending on the
- the pH is about 4.9 to about 5.2 with
- plant cell produces transiently expressed polypeptides, including proteins and enzymes, may
- Such monitoring includes optical density, pH, temperature, media nutrient levels including dissolved oxygen, and
- length of time of reaction is about 24 hours to about 4 days.
- the length of time is about 24 hours to about 4 days.
- the length of time is about 24 hours to about 4 days.
- the length of time is about 2 days.
- bacteria is added to the plant culture or mix. Although the time when the bacterial culture is not
- plant cell concentration a plant cell concentration, growth rate, nutrient levels, medium osmoticum, and/or medium
- a bacterial culture is added at about 7 to about 14 days of batch plant
- the point of addition of the bacterial culture is about 8 days of
- Plant tissue could also be produced in continuous culture to
- auxotroph means a culture of bacteria which cannot grow
- the auxotroph lacks the ability to metabolize amino acids
- vitamins such as thiamine and/or nucleic acid precursors such as purines and pyrimidines,
- auxotrophic since an organism can be multiply auxotrophic. More preferably, the auxotroph does not have
- auxotrophic mutants used that may not necessarily be limited to auxotrophic mutants, so long as the trait is such
- Agrobacterium requires certain external source of nutrients or stimuli for it to
- the present invention is also directed to achieving enhanced transient protein
- the present invention is further directed to monitoring and controlling plant cell and
- tissue culture growth to provide precise optimal timing of transient expression.
- root culture is used as a platform for quantitative transient gene
- biomass fresh and dry weight based on media measurements of inorganics e.g.
- carbohydrate e.g. refractive index
- tissue water content via medium
- dissolved oxygen levels can also be monitored.
- both root and cell suspension cultures are used to facilitate
- Agrobacterium tumefaciens is one of the primary vectors used in plant biotechnology
- Agrobacterium can transfer genes from its tumor-inducing (TI) plasmid (or RI plasmid for A. rhizogenes) to the TI plasmid (or RI plasmid for A. rhizogenes) to the TI plasmid (or RI plasmid for A. rhizogenes) to the TI plasmid (or RI plasmid for A. rhizogenes) to the tumor-inducing (TI) plasmid (or RI plasmid for A. rhizogenes) to the TI plasmid (or RI plasmid for A. rhizogenes) to the TI plasmid (or RI plasmid for A. rhizogenes) to the TI plasmid (or RI plasmid for A. rhizogenes) to the TI plasmid (or RI plasmid for A.
- T-DNA The genes encoded on the DNA that is transferred (T-DNA) can be
- protoplasts for transient expression including ballistics, polyethylene glycol (PEG) and
- Agrobacterium can accomplish transient expression without the need to
- Agrobacterium containing the transgene of interest is
- NLS's nuclear localization signals
- ScT SV40 T-antigen consensus NLS
- the M9-NLS complex with ScT will enhance direct plasmid expression
- refractive index as a measure of sugar concentration
- the plant biomass is normally greatest at the end of a bioreactor
- an auxotrophic bacterial mutant may be made, which will be subject to growth
- Root culture may have advantages over cell suspension cultures as a transient
- root culture is their superior genetic stability as compared to cell suspension cultures. This
- modification of the protein chemistry such as altered post-translational modification such as
- the plant host tissue need not be a cultured sample. Rather, it is
- Agrobacterium can be used to deliver a self-replicating viral construct
- Transient protein expression also may
- TMV transglutaminase
- simple virus addition or an Agro-
- Gemini viruses which are DNA viruses, also may be used
- composition and flow rate to achieve appropriate conditions for growth and production.
- the method of the invention incorporates a water balance into an overall mass balance
- enzyme activity is detected only after this intron is spliced out by the plant and expressed.
- FIG. 3 shows the level of plant-expressed GUS at different times after the addition of
- Equally important is the time at which the bacteria is added into the plant culture, as
- auxotrophs (imperative for 1000 L scale work) by using an Agrobacterium auxotroph.
- auxotrophs in FIG. 4 were generated by randomly inserting
- Auxotrophic Agrobacterium were added to cultures of actively growing Nicotiana
- FIG. 4 shows that (A)
- control is dependent on the auxotroph type, and (C) control is dependent on whether the co-
- cysteine auxotroph is found to work well for roots, but not cells, the pantothenate
- the glutamine/glutamate auxotroph works well for cells but not roots, the glutamine/glutamate auxotroph achieves control for both roots and cells, whereas the thiamine auxotroph was not controlled in either
- the level of GUS expression depends upon the physical environment. The
- tissue wounding shear stress
- FIG. 5 shows a graph of plant-expressed GUS
- FIG. 6 shows that increasing the time cells are subjected to wounding cause them to
- FIG. 7 shows a very interesting and novel twist to making this transient expression
- the inventors developed a way of making Hyoscyamus muticus plant roots
- FIG. 8 shows that the pH of the surrounding medium has a large effect on expression.
- compounds can be added to the media to simulate the wounded plant and
- acetosyringone is shown to be highly dependent upon the medium pH.
- Other phenolic / plant wounding based activators, such as syringaldehyde also may be used, as demonstrated
- Dissolved oxygen (DO) will be measured off-line by capturing
- biomass media and biomass composition
- total biomass
- corn endosperm culture also may be desirable for a variety of desirable dicot cultures such as tobacco.
- Corn endosperm cultures are of particular interest because they may confer unique
- Corn endosperm cultures display endosperm-specific metabolism such as zein
- bioreactor The operational strategy is as follows:
- Plant root cultures have no minimum inoculation concentration and can be
- V VM volume of culture per minute
- the culture will be accomplished with oxygen supplementation.
- RI refractive index
- Example 6 The principles set forth in Example 6 permit a precise calculation of the status of the
- bioreactors can be found in PCT/US99/19662 (WO 00/11953). This ability to systematically scale-up plant tissue culture permits systematic scale-up of the subsequent Agrobacterium co-
- Controlling the timing of product formation is important to achieve high titers of the
- Agrobacterium is activated for about 24 hours with about 100
- the 'induction' time course provides quantitative information on: the sensitivity of
- the experimental space covers a wide range of
- Plant cells display strikingly different morphologies in terms of degree of aggregation
- Agrobacterium infection as a batch culture. Based on typical protocols for infection, infection
- biomass loadings should favor higher productivities, the issues of phase of plant cell growth and
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Abstract
L'invention concerne un procédé destiné à produire de manière recombinante et transitoire un polypeptide dans un tissu végétal, qui consiste à présenter un échantillon de tissu végétal dans un bioréacteur, à ajouter audit échantillon, un échantillon d'agrobactérie renfermant une séquence nucléotidique codant le polypeptide sur l'ADN-T, à co-cultiver l'échantillon de tissu végétal avec l'agrobactérie de façon à transférer la séquence nucléotidique au végétal, le tissu végétal exprimant alors de manière transitoire le polypeptide puis séparant du mélange ledit polypeptide.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU76036/00A AU7603600A (en) | 1999-09-22 | 2000-09-22 | Quantitative transient protein expression in plant tissue culture |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US15532799P | 1999-09-22 | 1999-09-22 | |
US60/155,327 | 1999-09-22 |
Publications (1)
Publication Number | Publication Date |
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WO2001020974A1 true WO2001020974A1 (fr) | 2001-03-29 |
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ID=22554994
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/US2000/026011 WO2001020974A1 (fr) | 1999-09-22 | 2000-09-22 | Expression quantitative de proteines transitoire dans une culture de tissu vegetal |
Country Status (2)
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AU (1) | AU7603600A (fr) |
WO (1) | WO2001020974A1 (fr) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005086667A2 (fr) | 2004-02-27 | 2005-09-22 | The Dow Chemical Company | Production de peptides dans des cellules de plantes avec un rendement eleve |
EP1596803A2 (fr) * | 2002-12-23 | 2005-11-23 | Sunol Molecular Corporation | Production transitoire de proteines importantes au plan pharmaceutique dans des plantes |
EP2431458A1 (fr) * | 2005-06-28 | 2012-03-21 | Ventria Bioscience | Composants de milieu de culture cellulaire produits par des cellules végétales |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5677474A (en) * | 1988-07-29 | 1997-10-14 | Washington University | Producing commercially valuable polypeptides with genetically transformed endosperm tissue |
WO1998042857A1 (fr) * | 1997-03-26 | 1998-10-01 | Japan As Represented By Director General Of Ministry Of Agriculture, Forestry And Fisheries National Institute Of Agrobiological Resources | Procede d'introduction genique |
US5889190A (en) * | 1988-02-26 | 1999-03-30 | Biosource Technologies, Inc. | Recombinant plant viral nucleic acids |
US5948955A (en) * | 1991-07-13 | 1999-09-07 | International Flower Developments Pty Ltd | Transgenic plants having altered anthocyann levels |
-
2000
- 2000-09-22 WO PCT/US2000/026011 patent/WO2001020974A1/fr active Application Filing
- 2000-09-22 AU AU76036/00A patent/AU7603600A/en not_active Abandoned
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5889190A (en) * | 1988-02-26 | 1999-03-30 | Biosource Technologies, Inc. | Recombinant plant viral nucleic acids |
US5677474A (en) * | 1988-07-29 | 1997-10-14 | Washington University | Producing commercially valuable polypeptides with genetically transformed endosperm tissue |
US5948955A (en) * | 1991-07-13 | 1999-09-07 | International Flower Developments Pty Ltd | Transgenic plants having altered anthocyann levels |
WO1998042857A1 (fr) * | 1997-03-26 | 1998-10-01 | Japan As Represented By Director General Of Ministry Of Agriculture, Forestry And Fisheries National Institute Of Agrobiological Resources | Procede d'introduction genique |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1596803A2 (fr) * | 2002-12-23 | 2005-11-23 | Sunol Molecular Corporation | Production transitoire de proteines importantes au plan pharmaceutique dans des plantes |
EP1596803A4 (fr) * | 2002-12-23 | 2007-12-12 | Altor Bioscience Corp | Production transitoire de proteines importantes au plan pharmaceutique dans des plantes |
WO2005086667A2 (fr) | 2004-02-27 | 2005-09-22 | The Dow Chemical Company | Production de peptides dans des cellules de plantes avec un rendement eleve |
EP3388521A1 (fr) | 2004-02-27 | 2018-10-17 | Dow AgroSciences LLC | Production de peptides dans des cellules de plantes avec un rendement eleve |
EP2431458A1 (fr) * | 2005-06-28 | 2012-03-21 | Ventria Bioscience | Composants de milieu de culture cellulaire produits par des cellules végétales |
Also Published As
Publication number | Publication date |
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AU7603600A (en) | 2001-04-24 |
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