EP1100915A2 - Fibrous proteins and the production thereof - Google Patents

Fibrous proteins and the production thereof

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Publication number
EP1100915A2
EP1100915A2 EP99950481A EP99950481A EP1100915A2 EP 1100915 A2 EP1100915 A2 EP 1100915A2 EP 99950481 A EP99950481 A EP 99950481A EP 99950481 A EP99950481 A EP 99950481A EP 1100915 A2 EP1100915 A2 EP 1100915A2
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European Patent Office
Prior art keywords
protein
fiber protein
plant cell
plant
precursor fiber
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EP99950481A
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German (de)
French (fr)
Inventor
Klaus Düring
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MPB Cologne GmbH Molecular Plant und Protein Biotechnology
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MPB Cologne GmbH Molecular Plant und Protein Biotechnology
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Publication of EP1100915A2 publication Critical patent/EP1100915A2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • 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

Definitions

  • the present invention relates to a process for the production of fiber proteins in plant cells, plant cells which can be used for this purpose and fiber proteins obtained by the process.
  • Fiber proteins are proteins that have mechanical stability, e.g. Elasticity. They arise from precursor fiber proteins that undergo polymerization or cross-linking. This requires the presence of repetitive amino acid sequences in the precursor fiber proteins and the action of proteins which process the precursor fiber proteins. Fiber proteins are found in animal and human cells. Examples of fiber proteins are collagen and elastin. Both are components of connective tissue, e.g. Skin, tendons, ligaments and blood vessels. Collagen is created through cross-linking of tropocollagen molecules, while elastin is formed through cross-linking of tropoelastin molecules.
  • Fiber proteins are used for medical and cosmetic purposes. For this purpose, they are often isolated from animal cells. This poses a great risk, since diseases of the animals, e.g. BSE to which people can be transmitted.
  • the present invention is based on the applicant's knowledge that precursor fiber proteins can be produced in plant cells, which can then be treated with them.
  • processing proteins can be converted into the corresponding fiber proteins.
  • the production of precursor fiber proteins can take place in individual plant cells as well as in plants.
  • the conversion of precursor fiber proteins into the corresponding fiber proteins can take place in vitro as well as in vivo. In the latter case, this can be done, for example, by expressing the precursor fiber protein together with the protein processing it in a plant cell.
  • the applicant has made his findings on individual plant cells as well as on plants, in particular the potato plant.
  • fiber protein encompasses a fiber protein of any kind and origin. It can have a two- or three-dimensionally networked structure. It can also be an animal or human fiber protein. It can also be in wild type or modified form. The latter comprises a fiber protein whose amino acid sequence has been changed at one or more locations compared to the wild-type sequence. Such changes can be additions, substitutions, deletions and / or inversions of one or more amino acids. In particular, amino acids can be present, which are preferably expressed in plant cells. Furthermore, the fiber protein can be a fusion protein, the fusion partner being, for example, oleosin. This protein then enables the localization of the fiber protein in the oil phase of plant propagation material.
  • Fiber proteins that are in a modified form have a mechanical stability, for example elasticity, that at least matches that of the wild-type form is comparable.
  • Preferred fiber proteins are collagen and elastin and derivatives or fragments thereof. The above explanations apply accordingly to a changed form.
  • the expression “expression of a precursor fiber protein” encompasses any expression of a gene coding for a precursor fiber protein in a plant cell, the precursor fiber protein being able to be converted into the corresponding fiber protein in a customary manner, for example by crosslinking or polymerization.
  • the above statements regarding the expression “fiber protein” apply accordingly.
  • the precursor fiber protein can be present with or without a signal peptide.
  • the former can be the natural or a foreign signal peptide, for example, whereby an extracellular localization of the precursor fiber protein is achieved. In the latter, on the other hand, the precursor fiber protein is localized in the cytoplasm.
  • the precursor fiber protein can have a control peptide, as a result of which localization of the precursor fiber protein in certain compartments of the plant cell, for example ER, chloroplasts or vacuoles, is achieved.
  • Preferred precursor fiber proteins are tropocollagen and tropoelastin and derivatives or fragments thereof.
  • Conventional plant cell expression vectors can be used to express a gene encoding a precursor fiber protein. Such include regulatory elements, such as enhancer, promoter and termination sequences, which are recognized in plant cells. Examples of this are CaMV 35S promoter and termination sequences (cf. Odell, JT et al., Nature 313 (1985), 810-812).
  • the expression vectors can also contain selection markers, for example a neomycin or kanamycin resistance gene.
  • the expression vectors can contain sequences which promote their introduction into plant cells.
  • the expression vectors T-DNA can contain binary vectors such as pSR 8-30 or pSR 8-35 / 1 if they are to be introduced into plants via Agrobacterium tumefaciens (cf. Düring, K. et al., Plant Journal 3 ( 1993), 587-598; Porsch, P. et al., Plant Molecular Biology 37 (581-585, 1998).
  • the expression vectors can also be generated by methods . are introduced into plant cells for which they do not require any special sequences. Such methods are, for example, microinjection, electroporation, DNA transfer using polyethylene glycol, liposome fusion or particle gun.
  • plant cell includes plant cells of any kind and lineage. It can be single plant cells, freshly isolated or established as a cell line, or those that are present in a bandage. The latter is e.g. a plant or part of it. Examples of plants are monocotyledonous plants, such as corn, rice, wheat, barley and sugar cane, and dicotyledonous plants, such as potato, tobacco, tomato, tea, coffee, Brassicaceaen, in particular rapeseed and cabbage, and legumes, in particular pea, phaseolus, vicia and Soybean.
  • monocotyledonous plants such as corn, rice, wheat, barley and sugar cane
  • dicotyledonous plants such as potato, tobacco, tomato, tea, coffee, Brassicaceaen, in particular rapeseed and cabbage, and legumes, in particular pea, phaseolus, vicia and Soybean.
  • precursor fiber protein processing protein includes any protein that a precursor fiber protein can convert to the corresponding fiber protein.
  • the transfer can be carried out in the usual manner, e.g. B. by crosslinking or polymerization.
  • a protein examples of such a protein are lysine oxidases. It can also be proteinases that e.g. have been described in the case of collagen. The lysine oxidases or proteinases can be present as such or as derivatives or fragments thereof. The above explanations apply accordingly to a modified form of a fiber protein.
  • the expression "incubation of a precursor fiber protein with a protein processing it” includes any incubation of these proteins by means of which the precursor fiber protein can be converted into the corresponding fiber protein.
  • the incubation can take place, for example, in vitro.
  • Incubation can also take place in vivo.
  • the expression of both proteins can take place in different plant cells, which are then combined, whereby the precursor fiber protein is incubated with the protein processing it.
  • the expression of the precursor fiber protein and the protein processing it can also take place in the same plant cell.
  • the incubation of both proteins inevitably takes place in this plant cell.
  • Another object of the present invention is a plant cell that expresses a precursor fiber protein and a protein processing it.
  • a plant cell is also preferred which only expresses the latter of these proteins.
  • plant cell precursor fiber protein” and “precursor fiber protein processing protein”
  • the plant cell can also be in the form of a propagation material.
  • a plant according to the invention which expresses a precursor fiber protein, for example tropoelastin, and a protein which processes it, for example lysine oxidase.
  • a precursor fiber protein for example tropoelastin
  • a protein which processes it for example lysine oxidase.
  • the same can be done with a cDNA coding for a lysine oxidase.
  • the DNA molecules obtained are used to transform bacteria, for example E.
  • coli S17-1 which are suitable for transferring the DNA molecules into Agrobacterium tumefaciens, for example GV 3101.
  • Agrobacterium tumefaciens for example GV 3101.
  • E.coli ⁇ 17-1 and Agrobacterium tumefaciens GV 3101 are mixed together and incubated overnight.
  • Agrobacteria which ingested the DNA molecules are selected by growth on medium containing carbenicillin. These are then applied to the leaves of potato plants that have been cut off and carved several times on the midrib, and incubated for two days in the dark.
  • the agrobacteria are then removed and growth substances are added to the potato plants, so that shoots form. These are cut off and used to cultivate new potato plants.
  • the detection of the expression products tropoelastin and lysine oxidase or the elastin obtained is carried out by means of specific antibodies against these proteins. Reference is made to the examples below.
  • the present invention it is possible to produce fiber proteins in plant cells, in particular plants, in high purity.
  • the fiber proteins are therefore suitable for a wide variety of applications. Such are found e.g. in agriculture, chemistry, cosmetics manufacturing and medicine. In the latter case e.g. the use of fiber proteins for grafts and wound closures.
  • the fiber proteins are characterized in that they are free from animal or human viruses or pathogens.
  • the fiber proteins can be produced in huge quantities. This is particularly true when they are isolated from crops grown in fields. The present invention thus makes a great contribution to providing medicines safely and in large quantities.
  • Example 1 Production of elastin in potato plants
  • a cDNA for human elastin is used (see Fazio, MJ, Journal of Investigative Dermatology 91 (1988), 458-464).
  • This cDNA is provided with a PCR at the 5 'end with an Ncol and at the 3' end with an Xbal restriction site.
  • the cDNA fragment obtained is converted into the vector pRT 100, which contains an expression cassette with CaMV 35S promoter and termination sequences (cf. Töpfer, R. et al., Nucleic Acids Research 15 (1987), 5890; Odell, JT et al., Supra).
  • the expression cassette containing the elastin cDNA is isolated and inserted into the binary vector pSR 8-30 (cf. Düring, K. et al.; Porsch, P. et al., Above).
  • the expression vector pSR 8-30 elastin is obtained.
  • a cDNA for human lysine oxidase is also used (cf. Häffleläinen, E.R., Genomics 11 (1991), 508-516). This is treated as above and inserted into the binary vector pSR 8-30. The expression vector pSR 8-30-lysine oxidase is obtained.
  • the expression vectors pSR 8-30-elastin and pSR 8-30-lysine oxidase are used to transform E. coli S17-1. Transformants are mixed with Agrobacterium tumefaciens GV 3101 and incubated overnight at 27 ° C (see Koncz, C, Shell, J., Molecular and General Genetics 204 (1986), 383-396; Koncz, C. et al., Proc Natl. Acad. Sci. USA 84 (1987), 131-135). It is selected for carbenicillin, the bla gene required for this being present in the above expression vectors.
  • Be ect i ons clones of Agrobacterium tumefaciens are cut on leaves of the potato plant cv that have been cut off and scratched several times on the midrib. Desiree applied and the plant is incubated for 2 days at 20 ° C in the dark. The agrobacteria are then separated off and plant growth substances are added to the potato plant, so that shoots preferably form. Furthermore, by adding kanamycin to the plant medium, the cells of the potato plant are not killed. Growing shoots are cut off and rooted on medium without plant growth substances, but with kanamycin. The further cultivation of the potato plants is carried out in the usual way.
  • tropoelastin and lysine oxidase can be expressed in plant cells, in particular in a plant. Furthermore, it can be seen that the incubation of lysine oxidase with the tropoelastin converts the latter into elastin and this can be isolated in pure form.
  • CDNAs are used which code for the subunits ⁇ 1 and ⁇ 2 of the human tropocollagen (see Chu, ML et al., Journal of Biological Chemistry 260 (1985), 2315-2320; Dickson LA et al., Nucleic Acids Res. 13 (1985), 3427-3438). Furthermore, cDNAs are used which code for human lysine oxidase, human procollagen C-proteinase or procollagen N-proteinase from bovine (cf. Häffleläinen, ER et al., Supra; Li, SW et al., Proc. Natl. Acad. Sci. USA 93 (1996), 5127-5130; Colige, A. et al., Proc. Natl. Acad. Sci. USA 94 (1997), 2374-2379).
  • cDNAs are treated as described in Example 1 and inserted into the vector pSR 8-30.
  • the expression vectors pSR 8-30 tropocollagen ⁇ 1, pSR 8-30 tropocollagen ⁇ 2, pSR 8-30 lysine oxidase, pSR 8-30-C proteinase and pSR 8-30-N proteinase are obtained.
  • the further procedure is as described in Example 1.
  • tropocollagen and proteins processing it can be expressed in plant cells, in particular in a plant. It also shows that collagen can be obtained which has a high purity.

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Abstract

The present invention relates to a method for the production of a fibrous protein, comprising the following steps: (a) expression of a precursor fibrous protein in a plant cell and (b) incubation of the precursor fibrous protein with a protein processing the latter. The invention also relates to the plant cells used for this purpose and to the fibrous proteins produced according to the inventive method.

Description

Faserproteine und ihre Herstellung Fiber proteins and their production
Die vorliegende Erfindung betrifft ein Verfahren zur Herstellung von Faserproteinen in Pflanzenzellen, hierfür verwendbare Pflanzenzellen und durch das Verfahren erhaltene Faserproteine .The present invention relates to a process for the production of fiber proteins in plant cells, plant cells which can be used for this purpose and fiber proteins obtained by the process.
Faserproteine sind Proteine, die eine mechanische Stabilität, z.B. Elastizität, aufweisen. Sie entstehen aus Vorläufer- Faserproteinen, die eine Polymerisierung bzw. Quervernetzung erfahren. Hierfür ist das Vorliegen von repetetiven Aminosäuresequenzen in den Vorläufer-Faserproteinen und das Einwirken von Proteinen notwendig, die Vorläufer-Faserproteine prozessieren. Faserproteine finden sich in tierischen und menschlichen Zellen. Beispiele von Faserproteine sind Kollagen und Elastin. Beide sind Bestandteile von Bindegeweben, z.B. Haut, Sehnen, Bändern und Blutgefäßen. Kollagen entsteht durch Quervernetzung von Tropokollagen-Molekülen, während Elastin durch Quervernetzung von Tropoelastin-Molekülen gebildet wird.Fiber proteins are proteins that have mechanical stability, e.g. Elasticity. They arise from precursor fiber proteins that undergo polymerization or cross-linking. This requires the presence of repetitive amino acid sequences in the precursor fiber proteins and the action of proteins which process the precursor fiber proteins. Fiber proteins are found in animal and human cells. Examples of fiber proteins are collagen and elastin. Both are components of connective tissue, e.g. Skin, tendons, ligaments and blood vessels. Collagen is created through cross-linking of tropocollagen molecules, while elastin is formed through cross-linking of tropoelastin molecules.
Faserproteine werden für medizinische bzw. kosmetische Zwecke verwendet. Hierzu werden sie vielfach aus Zellen von Tieren isoliert. Dies bringt ein großes Risiko, da somit Erkrankungen der Tiere, z.B. BSE, auf den Menschen übertragen werden können.Fiber proteins are used for medical and cosmetic purposes. For this purpose, they are often isolated from animal cells. This poses a great risk, since diseases of the animals, e.g. BSE to which people can be transmitted.
Der vorliegenden Erfindung liegt daher die Aufgabe zugrunde, ein Verfahren bereitzustellen, mit dem Faserproteine ohne die vorstehenden Risiken hergestellt werden können.It is therefore the object of the present invention to provide a method with which fiber proteins can be produced without the above risks.
Erfindungsgemäß wird dies durch die Gegenstände in den Patentansprüchen erreicht.According to the invention, this is achieved by the subject matter in the claims.
Die vorliegende Erfindung beruht auf den Erkenntnissen des Anmelders, daß Vorläufer-Faserproteine in Pflanzenzellen hergestellt werden können, die dann durch Behandlung mit sie. prozessierenden Proteinen in die entsprechenden Faserproteine überführt werden können. Insbesondere hat er erkannt, daß die Herstellung von Vorläufer-Faserproteinen in einzelnen Pflanzenzellen wie auch in Pflanzen erfolgen kann. Ferner hat er erkannt, daß die Überführung von Vorläufer-Faserproteinen in die entsprechenden Faserproteine in vitro wie auch in vivo erfolgen kann. In letzterem Fall kann dies z.B. dadurch erfolgen, daß das Vorlaufer-Faserprotein zusammen mit dem es prozessierenden Protein in einer Pflanzenzelle exprimiert wird. Der Anmelder hat seine Erkenntnisse an einzelnen Pflanzenzellen wie auch an Pflanzen, insbesondere der Kartoffelpflanze, gemacht.The present invention is based on the applicant's knowledge that precursor fiber proteins can be produced in plant cells, which can then be treated with them. processing proteins can be converted into the corresponding fiber proteins. In particular, he recognized that the production of precursor fiber proteins can take place in individual plant cells as well as in plants. He also recognized that the conversion of precursor fiber proteins into the corresponding fiber proteins can take place in vitro as well as in vivo. In the latter case, this can be done, for example, by expressing the precursor fiber protein together with the protein processing it in a plant cell. The applicant has made his findings on individual plant cells as well as on plants, in particular the potato plant.
Erfindungsgemäß werden die Erkenntnisse des Anmelders für ein Verfahren zur Herstellung eines Faserproteins genutzt, das die folgenden Verfahrensschritte umfaßt:According to the invention, the knowledge of the applicant is used for a process for the production of a fiber protein, which comprises the following process steps:
(a) Expression eines Vorläufer-Faserproteins in einer Pflanzenzelle, und(a) expression of a precursor fiber protein in a plant cell, and
(b) Inkubation des Vorlaufer-Faserproteins mit einem es prozessierenden Protein.(b) Incubation of the precursor fiber protein with a protein processing it.
Der Ausdruck "Faserprotein" umfaßt ein Faserprotein jeglicher Art und Abstammung. Es kann eine zwei- oder dreidimensional vernetzte Struktur aufweisen. Auch kann es ein tierisches oder menschliches Faserprotein sein. Ferner kann es in Wildtypoder veränderter Form vorliegen. Letzteres umfaßt ein Faserprotein dessen Aminosäuresequenz im Vergleich zur Wildtyp- Sequenz an ein oder mehreren Stellen verändert ist. Solche Veränderungen können Additionen, Substitutionen, Deletionen und/oder Inversionen von ein oder mehreren Aminosäuren sein. Insbesondere können Aminosäuren vorliegen, die in Pflanzenzellen bevorzugt exprimiert werden. Ferner kann das Faserprotein ein Fusionsprotein sein, wobei der Fusionspartner z.B. Oleosin sein kann. Dieses Protein ermöglicht dann die Lokalisation des Faserproteins in der Ölphase von pflanzlichem Vermehrungsmaterial. Faserproteine, die in veränderter Form vorliegen, weisen eine mechanische Stabilität, z.B. Elastizität, auf, die zumindest mit jener der Wildtyp-Form vergleichbar ist. Bevorzugte Faserproteine sind Kollagen und Elastin sowie Derivate bzw. Fragmente davon. Für sie gelten vorstehende Ausführungen hinsichtlich einer veränderten Form entsprechend.The term "fiber protein" encompasses a fiber protein of any kind and origin. It can have a two- or three-dimensionally networked structure. It can also be an animal or human fiber protein. It can also be in wild type or modified form. The latter comprises a fiber protein whose amino acid sequence has been changed at one or more locations compared to the wild-type sequence. Such changes can be additions, substitutions, deletions and / or inversions of one or more amino acids. In particular, amino acids can be present, which are preferably expressed in plant cells. Furthermore, the fiber protein can be a fusion protein, the fusion partner being, for example, oleosin. This protein then enables the localization of the fiber protein in the oil phase of plant propagation material. Fiber proteins that are in a modified form have a mechanical stability, for example elasticity, that at least matches that of the wild-type form is comparable. Preferred fiber proteins are collagen and elastin and derivatives or fragments thereof. The above explanations apply accordingly to a changed form.
Der Ausdruck "Expression eines Vorlaufer-Faserproteins" umfaßt jegliche Expression eines für ein Vorläufer-Faserprotein kodierenden Gens in einer Pflanzenzelle, wobei das Vorläufer- Faserprotein in üblicher Weise, z.B. durch Quervernetzung oder Polymerisierung, in das entsprechende Faserprotein überführt werden kann. Vorstehende Ausführungen zu dem Ausdruck "Faserprotein" gelten hier entsprechend. Ferner kann das Vorläufer-Faserprotein mit oder ohne Signalpeptid vorliegen. Ersteres kann z.B. das natürliche oder ein fremdes Signalpeptid sein, wodurch eine extrazelluläre Lokalisation des Vorläufer-Faserproteins erreicht wird. Bei letzterem wird dagegen eine Lokalisation des Vorläufer-Faserproteins im Zytoplasma erreicht. Deweiteren kann das Vorläufer-Faserprotein ein Steuerpeptid aufweisen, wodurch eine Lokalisation des Vorläufer-Faserproteins in bestimmten Kompartimenten der Pflanzenzelle, z.B. ER, Chloroplasten oder Vakuolen, erreicht wird. Bevorzugte Vorläufer-Faserproteine sind Tropokollagen und Tropoelastin sowie Derivate bzw. Fragmente davon. Für die Expression eines für ein Vorläufer-Faserprotein kodierenden Gens können übliche Expressionsvektoren für Pflanzenzellen verwendet werden. Solche umfassen Regulationselemente, z.B. Enhancer- , Promotor- und Terminations-Sequenzen, die in Pflanzenzellen erkannt werden. Beispiele hierfür sind CaMV 35S Promotor- und Terminations-Sequenzen (vgl. Odell, J. T. et al., Nature 313 (1985), 810-812) . Auch können die Expressionsvektoren, Selektionsmarker, z.B. ein Neomycin- oder Kanamycin-Resistenz-Gen, enthalten. Desweiteren können die Expressionsvektoren Sequenzen enthalten, die ihre Einschleusung in Pflanzenzellen begünstigen. Beispielsweise können die Expressionsvektoren T-DNA von binären Vektoren, wie pSR 8-30 oder pSR 8-35/1 enthalten, wenn sie über Agrobacterium tumefaciens in Pflanzen eingeschleust werden sollen (vgl. Düring, K. et al . , Plant Journal 3 (1993), 587-598; Porsch, P. et al., Plant Molecular Biology 37 (1998), 581-585). Darüber hinaus können die Expressionsvektoren auch durch Verfahren . in Pflanzenzellen eingeschleust werden, für die sie keine speziellen Sequenzen benötigen. Solche Verfahren sind z.B. Mikroinjektion, Elektroporation, DNA-Transfer mittels Polyethylenglykol, Liposomenfusion oder Partikelkanone.The expression “expression of a precursor fiber protein” encompasses any expression of a gene coding for a precursor fiber protein in a plant cell, the precursor fiber protein being able to be converted into the corresponding fiber protein in a customary manner, for example by crosslinking or polymerization. The above statements regarding the expression “fiber protein” apply accordingly. Furthermore, the precursor fiber protein can be present with or without a signal peptide. The former can be the natural or a foreign signal peptide, for example, whereby an extracellular localization of the precursor fiber protein is achieved. In the latter, on the other hand, the precursor fiber protein is localized in the cytoplasm. Furthermore, the precursor fiber protein can have a control peptide, as a result of which localization of the precursor fiber protein in certain compartments of the plant cell, for example ER, chloroplasts or vacuoles, is achieved. Preferred precursor fiber proteins are tropocollagen and tropoelastin and derivatives or fragments thereof. Conventional plant cell expression vectors can be used to express a gene encoding a precursor fiber protein. Such include regulatory elements, such as enhancer, promoter and termination sequences, which are recognized in plant cells. Examples of this are CaMV 35S promoter and termination sequences (cf. Odell, JT et al., Nature 313 (1985), 810-812). The expression vectors can also contain selection markers, for example a neomycin or kanamycin resistance gene. Furthermore, the expression vectors can contain sequences which promote their introduction into plant cells. For example, the expression vectors T-DNA can contain binary vectors such as pSR 8-30 or pSR 8-35 / 1 if they are to be introduced into plants via Agrobacterium tumefaciens (cf. Düring, K. et al., Plant Journal 3 ( 1993), 587-598; Porsch, P. et al., Plant Molecular Biology 37 (581-585, 1998). In addition, the expression vectors can also be generated by methods . are introduced into plant cells for which they do not require any special sequences. Such methods are, for example, microinjection, electroporation, DNA transfer using polyethylene glycol, liposome fusion or particle gun.
Der Ausdruck "Pflanzenzelle" umfaßt Pflanzenzellen jeglicher Art und Abstammung. Es können einzelne Pflanzenzellen, frisch isoliert oder als Zellinie etabliert, oder solche sein, die in einem Verband vorliegen. Letzteres ist z.B. eine Pflanze oder ein Teil davon. Beispiele von Pflanzen sind monocotyle Pflanzen, wie Mais, Reis, Weizen, Gerste und Zuckerrohr, und dicotyle Pflanzen, wie Kartoffel, Tabak, Tomate, Tee, Kaffee, Brassicaceaen, insbesondere Raps und Kohl, und Leguminosen, insbesondere Erbse, Phaseolus, Vicia und Sojabohne.The term "plant cell" includes plant cells of any kind and lineage. It can be single plant cells, freshly isolated or established as a cell line, or those that are present in a bandage. The latter is e.g. a plant or part of it. Examples of plants are monocotyledonous plants, such as corn, rice, wheat, barley and sugar cane, and dicotyledonous plants, such as potato, tobacco, tomato, tea, coffee, Brassicaceaen, in particular rapeseed and cabbage, and legumes, in particular pea, phaseolus, vicia and Soybean.
Der Ausdruck "Vorläufer-Faserprotein prozessierendes Protein" umfaßt jegliches Protein, das ein Vorläufer-Faserprotein in das entsprechende Faserprotein überführen kann. Die Überführung kann in üblicher Weise, z. B. durch Quervernetzung oder Polymerisierung, erfolgen. Beispiele eines solchen Proteins sind Lysinoxidasen. Auch können es Proteinasen sein, die z.B. im Falle von Kollagen beschrieben worden sind. Die Lysinoxidasen bzw. Proteinasen können als solche bzw. als Derivate oder Fragmente davon vorliegen. Für sie gelten vorstehende Ausführungen hinsichtlich einer veränderten Form eines Faserproteins entsprechend.The term "precursor fiber protein processing protein" includes any protein that a precursor fiber protein can convert to the corresponding fiber protein. The transfer can be carried out in the usual manner, e.g. B. by crosslinking or polymerization. Examples of such a protein are lysine oxidases. It can also be proteinases that e.g. have been described in the case of collagen. The lysine oxidases or proteinases can be present as such or as derivatives or fragments thereof. The above explanations apply accordingly to a modified form of a fiber protein.
Der Ausdruck "Inkubation eines Vorläufer-Faserproteins mit einem es prozessierenden Protein" umfaßt jegliche Inkubation dieser Proteine, durch die das Vorläufer-Faserprotein in das entsprechende Faserprotein überführt werden kann. Die Inkubation kann z.B. in vitro erfolgen. Hierzu ist es günstig, das exprimierte Vorläufer-Faserprotein in Lösung mit dem es prozessierenden Protein zu inkubieren. Auch kann die Inkubation in vivo erfolgen. Hierzu ist es günstig, nicht nur das Vorläufer-Faserprotein, sondern auch das es prozessierende Protein in einer Pflanzenzelle zu exprimieren. Die Expression beider Proteine kann in verschiedenen Pflanzenzellen erfolgen, die dann vereinigt werden, wodurch eine Inkubation des Vorläufer-Faserproteins mit dem es prozessierenden Proteins erfolgt. Auch kann die Expression des Vorläufer-Faserproteins und des es prozessierenden Proteins in der gleichen Pflanzenzelle erfolgen. Die Inkubation beider Proteine erfolgt damit zwangsläufig in dieser Pflanzenzelle. Für die Expression eines ein Vorläufer-Faserprotein prozessierenden Proteins gelten vorstehende Ausführungen hinsichtlich der Expression eines Vorläufer-Faserproteins entsprechend.The expression "incubation of a precursor fiber protein with a protein processing it" includes any incubation of these proteins by means of which the precursor fiber protein can be converted into the corresponding fiber protein. The incubation can take place, for example, in vitro. For this purpose, it is expedient to incubate the expressed precursor fiber protein in solution with the protein processing it. Incubation can also take place in vivo. For this purpose, it is beneficial not only for the precursor fiber protein, but also for the one that processes it To express protein in a plant cell. The expression of both proteins can take place in different plant cells, which are then combined, whereby the precursor fiber protein is incubated with the protein processing it. The expression of the precursor fiber protein and the protein processing it can also take place in the same plant cell. The incubation of both proteins inevitably takes place in this plant cell. The above statements regarding the expression of a precursor fiber protein apply correspondingly to the expression of a protein processing a precursor fiber protein.
Ein weiterer Gegenstand der vorliegenden Erfindung ist eine Pflanzenzelle, die ein Vorläufer-Faserprotein und ein es prozessierendes Protein exprimiert. Auch ist eine Pflanzenzelle bevorzugt, die von diesen Proteinen nur letzteres exprimiert. Hinsichtlich der Ausdrücke "Pflanzenzelle", "Vorläufer-Faserprotein" und "Vorläufer- Faserprotein prozessierendes Protein" wird auf vorstehende Ausführungen verwiesen. Ferner kann die Pflanzenzelle in Form eines Vermehrungsmaterials vorliegen.Another object of the present invention is a plant cell that expresses a precursor fiber protein and a protein processing it. A plant cell is also preferred which only expresses the latter of these proteins. With regard to the terms “plant cell”, “precursor fiber protein” and “precursor fiber protein processing protein”, reference is made to the above statements. The plant cell can also be in the form of a propagation material.
Zur Herstellung einer erfindungsgemäßen Pflanzenzelle können übliche Verfahren verwendet werden. Ergänzend zu vorstehenden Ausführungen wird beispielhaft die Herstellung einer erfindungsgemäßen Pflanze beschrieben, die ein Vorläufer- Faserprotein, z.B. Tropoelastin und ein es prozessierendes Protein, z.B. Lysinoxidase, exprimiert. Hierzu ist es günstig, eine für Tropoelastin kodierende cDNA mit CaMV 35S Promotor- und Terminations-Sequenzen zu versehen und in einen binären Vektor, z.B. pSR 8-30 bzw. pSR 8-35/1 zu inserieren. Gleiches kann mit einer für eine Lysinoxidase kodierende cDNA erfolgen. Die erhaltenen DNA-Moleküle werden zur Transformation von Bakterien, z.B. E.coli S17-1 verwendet, die sich für einen Transfer der DNA-Moleküle in Agrobacterium tumefaciens, z.B. GV 3101, eignen. Hierzu werden E.coli Ξ17-1 und Agrobakterium tumefaciens GV 3101 miteinander gemischt und über Nacht inkubiert. Agrobakterien, welche die DNA-Moleküle aufgenommen haben, werden durch Wachstum auf Carbenicillin-haltigem Medium selektioniert . Diese werden dann auf abgeschnittenen und mehrfach an der Mittelrippe eingeritzten Blättern von Kartoffelpflanzen aufgebracht und zwei Tage im Dunkeln inkubiert. Danach werden die Agrobakterien entfernt und den Kartoffelpflanzen Wuchsstoffe zugegeben, so daß sich Sprosse ausbilden. Diese werden abgeschnitten und zur Kultivierung von neuen Kartoffelpflanzen verwendet. Der Nachweis der Expressionsprodukte Tropoelastin und Lysinoxidase bzw. des erhaltenen Elastins wird mittels spezifischer Antikörper gegen diese Proteine durchgeführt. Es wird auf die nachfolgenden Beispiele verwiesen.Conventional methods can be used to produce a plant cell according to the invention. In addition to the above, the production of a plant according to the invention is described by way of example, which expresses a precursor fiber protein, for example tropoelastin, and a protein which processes it, for example lysine oxidase. For this purpose, it is favorable to provide a cDNA coding for tropoelastin with CaMV 35S promoter and termination sequences and to insert it in a binary vector, for example pSR 8-30 or pSR 8-35 / 1. The same can be done with a cDNA coding for a lysine oxidase. The DNA molecules obtained are used to transform bacteria, for example E. coli S17-1, which are suitable for transferring the DNA molecules into Agrobacterium tumefaciens, for example GV 3101. For this purpose, E.coli Ξ17-1 and Agrobacterium tumefaciens GV 3101 are mixed together and incubated overnight. Agrobacteria, which ingested the DNA molecules are selected by growth on medium containing carbenicillin. These are then applied to the leaves of potato plants that have been cut off and carved several times on the midrib, and incubated for two days in the dark. The agrobacteria are then removed and growth substances are added to the potato plants, so that shoots form. These are cut off and used to cultivate new potato plants. The detection of the expression products tropoelastin and lysine oxidase or the elastin obtained is carried out by means of specific antibodies against these proteins. Reference is made to the examples below.
Mit der vorliegenden Erfindung ist es möglich, Faserproteine in Pflanzenzellen, insbesondere Pflanzen, in großer Reinheit herzustellen. Die Faserproteine eignen sich daher für die verschiedensten Anwendungen. Solche finden sich z.B. in der Landwirtschaft, Chemie, Kosmetik-Herstellung und Medizin. In letzterem Fall ist z.B. der Einsatz von Faserproteinen für Transplantate und Wundverschlüsse zu nennen. Insbesondere zeichnen sich die Faserproteine dadurch aus, daß sie frei von tierischen oder menschlichen Viren bzw. Krankheitserregern sind. Deweiteren können die Faserproteine in rießigen Mengen hergestellte werden. Dies trifft insbesondere zu, wenn sie aus auf Feldern kultivierten Pflanzen isoliert werden. Die vorliegende Erfindung stellt somit einen großen Beitrag dar, Arzneimittel sicher und in großen Mengen bereitzustellen.With the present invention it is possible to produce fiber proteins in plant cells, in particular plants, in high purity. The fiber proteins are therefore suitable for a wide variety of applications. Such are found e.g. in agriculture, chemistry, cosmetics manufacturing and medicine. In the latter case e.g. the use of fiber proteins for grafts and wound closures. In particular, the fiber proteins are characterized in that they are free from animal or human viruses or pathogens. Furthermore, the fiber proteins can be produced in huge quantities. This is particularly true when they are isolated from crops grown in fields. The present invention thus makes a great contribution to providing medicines safely and in large quantities.
Die Erfindung wird durch die nachfolgenden Beispiele erläutert .The invention is illustrated by the following examples.
Beispiel 1: Herstellung von Elastin in KartoffelpflanzenExample 1: Production of elastin in potato plants
Es wird eine cDNA für humanes Elastin verwendet (vgl. Fazio, M.J., Journal of Investigative Dermatology 91 (1988), 458- 464). Diese cDNA wird mittels einer PCR am 5 ' -Ende mit einer Ncol- und am 3 ' -Ende mit einer Xbal-Restriktionsstelle versehen. Das erhaltene cDNA-Fragment wird in den Vektor pRT 100 inseriert, der eine Expressionskassette mit CaMV 35S Promotor- und Terminations-Sequenzen enthält (vgl. Töpfer, R. et al., Nucleic Acids Research 15 (1987), 5890; Odell, J. T. et al., vorstehend). Nach Spaltung mit Hindlll wird die die Elastin-cDNA enthaltende Expressionskassette isoliert und in den binären Vektor pSR 8-30 inseriert (vgl. Düring, K. et al . ; Porsch, P. et al . , vorstehend). Es wird der Expressionsvektor pSR 8-30-Elastin erhalten.A cDNA for human elastin is used (see Fazio, MJ, Journal of Investigative Dermatology 91 (1988), 458-464). This cDNA is provided with a PCR at the 5 'end with an Ncol and at the 3' end with an Xbal restriction site. The cDNA fragment obtained is converted into the vector pRT 100, which contains an expression cassette with CaMV 35S promoter and termination sequences (cf. Töpfer, R. et al., Nucleic Acids Research 15 (1987), 5890; Odell, JT et al., Supra). After cleavage with HindIII, the expression cassette containing the elastin cDNA is isolated and inserted into the binary vector pSR 8-30 (cf. Düring, K. et al.; Porsch, P. et al., Above). The expression vector pSR 8-30 elastin is obtained.
Ferner wird eine cDNA für humane Lysinoxidase verwendet (vgl. Hämäläinen, E.R., Genomics 11 (1991), 508-516). Diese wird wie vorstehend behandelt und in den binären Vektor pSR 8-30 inseriert. Es wird der Expressionsvektor pSR 8-30-Lysinoxidase erhalten.A cDNA for human lysine oxidase is also used (cf. Hämäläinen, E.R., Genomics 11 (1991), 508-516). This is treated as above and inserted into the binary vector pSR 8-30. The expression vector pSR 8-30-lysine oxidase is obtained.
Die Expressionsvektoren pSR 8-30-Elastin und pSR 8-30- Lysinoxidase werden zur Transformation von E.coli S17-1 verwendet. Transformanten werden mit Agrobacterium tumefaciens GV 3101 gemischt und über Nacht bei 27°C inkubiert (vgl. Koncz, C, Shell, J., Molecular and General Genetics 204 (1986), 383- 396; Koncz, C. et al . , Proc . Natl . Acad. Sei. USA 84 (1987), 131-135) . Es wird auf Carbenicillin selektioniert , wobei das hierfür notwendige bla-Gen in den vorstehenden Expressionsvektoren vorliegt. Sei ekt i onsklone von Agrobacterium tumefaciens werden auf abgeschnittenen und mehrfach an der Mittelrippe eingeritzten Blättern der Kartoffelpflanze cv. Desiree aufgebracht und die Pflanze wird 2 Tage bei 20°C im Dunkeln inkubiert. Danach werden die Agrobakter ien abgetrennt und der Kartoffelpflanze Pflanzenwuchsstoffe zugesetzt, so daß sich bevorzugt Sprosse ausbilden. Ferner werden durch Zugabe von Kanamycin in das Pflanzenmedium ni ch t - t r ans f o rmi er t e Zellen der Kartoffelpflanze abgetötet. Heranwachsende Sprosse werden abgeschnitten und auf Medium ohne Pflanzenwachstu sstoffe, aber mit Kanamycin, bewurzelt. Die weitere Kultivierung der Kartoffelpflanzen erfolgt in üblicher Weise.The expression vectors pSR 8-30-elastin and pSR 8-30-lysine oxidase are used to transform E. coli S17-1. Transformants are mixed with Agrobacterium tumefaciens GV 3101 and incubated overnight at 27 ° C (see Koncz, C, Shell, J., Molecular and General Genetics 204 (1986), 383-396; Koncz, C. et al., Proc Natl. Acad. Sci. USA 84 (1987), 131-135). It is selected for carbenicillin, the bla gene required for this being present in the above expression vectors. Be ect i ons clones of Agrobacterium tumefaciens are cut on leaves of the potato plant cv that have been cut off and scratched several times on the midrib. Desiree applied and the plant is incubated for 2 days at 20 ° C in the dark. The agrobacteria are then separated off and plant growth substances are added to the potato plant, so that shoots preferably form. Furthermore, by adding kanamycin to the plant medium, the cells of the potato plant are not killed. Growing shoots are cut off and rooted on medium without plant growth substances, but with kanamycin. The further cultivation of the potato plants is carried out in the usual way.
Der Nachweis des exprimierten Tropoelas tins und der Lysinoxidase bzw. des erhaltenen Elastins wird durch Antikörper im Western Blot bzw. ELISA erreicht, die spezifisch für die einzelnen Proteine sind. Hierzu wird Gesamt-Protein oder die interzelluläre Waschflüssigkeit der Kartoffelpflanze isoliert und in die entsprechenden Nachweisverfahren eingesetzt .Evidence of the expressed tropoelastin and the Lysine oxidase or the elastin obtained is achieved by antibodies in the Western blot or ELISA, which are specific for the individual proteins. For this purpose, total protein or the intercellular washing liquid of the potato plant is isolated and used in the corresponding detection methods.
Es zeigt sich, daß Tropoelastin und Lysinoxidase in Pflanzenzellen, insbesondere in einer Pflanze, exprimiert werden können. Ferner zeigt sich, daß durch die Inkubation von Lysinoxidase mit dem Tropoelastin letzteres in Elastin überführt wird und dieses in reiner Form isoliert werden kann.It is shown that tropoelastin and lysine oxidase can be expressed in plant cells, in particular in a plant. Furthermore, it can be seen that the incubation of lysine oxidase with the tropoelastin converts the latter into elastin and this can be isolated in pure form.
Beispiel 2 : Herstellung von Kollagen in KartoffelpflanzenExample 2: Production of collagen in potato plants
Es werden cDNAs verwendet, die für die Untereinheiten αl und α2 des humanen Tropokollagens kodieren (vgl. Chu, M.L. et al . , Journal of Biological Chemistry 260 (1985), 2315-2320; Dickson L.A. et al., Nucleic Acids Res . 13 (1985), 3427-3438). Ferner werden cDNAs verwendet, die für humane Lysinoxidase, humane Prokollagen C-Proteinase bzw. Prokollagen N-Proteinase aus dem Rind kodieren (vgl. Hämäläinen, E.R. et al . , vorstehend; Li, S.W. et al., Proc. Natl . Acad. Sei. USA 93 (1996), 5127-5130; Colige, A. et al . , Proc. Natl. Acad. Sei. USA 94 (1997), 2374- 2379) .CDNAs are used which code for the subunits α1 and α2 of the human tropocollagen (see Chu, ML et al., Journal of Biological Chemistry 260 (1985), 2315-2320; Dickson LA et al., Nucleic Acids Res. 13 (1985), 3427-3438). Furthermore, cDNAs are used which code for human lysine oxidase, human procollagen C-proteinase or procollagen N-proteinase from bovine (cf. Hämäläinen, ER et al., Supra; Li, SW et al., Proc. Natl. Acad. Sci. USA 93 (1996), 5127-5130; Colige, A. et al., Proc. Natl. Acad. Sci. USA 94 (1997), 2374-2379).
Diese cDNAs werden, wie in Beispiel 1 beschrieben, behandelt und in den Vektor pSR 8-30 inseriert. Es werden die Expressionsvektoren pSR 8-30-Tropokollagen αl , pSR 8-30- Tropokollagen α2 , pSR 8-30-Lysinoxidase, pSR 8-30-C-Proteinase und pSR 8-30-N-Proteinase erhalten. Das weitere Vorgehen erfolgt entsprechend der Beschreibung von Beispiel 1.These cDNAs are treated as described in Example 1 and inserted into the vector pSR 8-30. The expression vectors pSR 8-30 tropocollagen α1, pSR 8-30 tropocollagen α2, pSR 8-30 lysine oxidase, pSR 8-30-C proteinase and pSR 8-30-N proteinase are obtained. The further procedure is as described in Example 1.
Es zeigt sich, daß Tropokollagen und es prozessierende Proteine in Pflanzenzellen, insbesondere in einer Pflanze, exprimiert werden können. Ferner zeigt sich, daß Kollagen erhalten werden kann, das eine hohe Reinheit aufweist. It is shown that tropocollagen and proteins processing it can be expressed in plant cells, in particular in a plant. It also shows that collagen can be obtained which has a high purity.

Claims

Patentansprüche claims
1. Verfahren zur Herstellung eines Faserproteins, umfassend die folgenden Verfahrensschritte:1. A process for producing a fiber protein, comprising the following process steps:
(a) Expression eines Vorläufer-Faserproteins in einer Pflanzenzelle, und(a) expression of a precursor fiber protein in a plant cell, and
(b) Inkubation des Vorläufer-Faserproteins mit einem es prozessierenden Protein.(b) Incubation of the precursor fiber protein with a protein processing it.
2. Verfahren nach Anspruch 1, wobei das prozessierende Protein in einer Pflanzenzelle exprimiert wird.2. The method of claim 1, wherein the processing protein is expressed in a plant cell.
3. Verfahren nach Anspruch 2, wobei die Expression des Vorläufer-Faserproteins und des es prozessierenden Proteins in verschiedenen Pflanzenzellen erfolgt.3. The method according to claim 2, wherein the expression of the precursor fiber protein and the protein processing it takes place in different plant cells.
4. Verfahren nach Anspruch 2, wobei die Expression des Vorläufer-Faserproteins und des es prozessierenden Proteins in der gleichen Pflanzenzelle erfolgt.4. The method according to claim 2, wherein the expression of the precursor fiber protein and the protein processing it takes place in the same plant cell.
5. Verfahren nach einem der Ansprüche 1 - 4, wobei die Pflanzenzelle in Form einer Pflanze vorliegt.5. The method according to any one of claims 1-4, wherein the plant cell is in the form of a plant.
6. Verfahren nach einem der Ansprüche 1 - 5 , wobei das Vorläufer-Faserprotein ein Prokollagen oder ein Derivat bzw. Fragment davon ist.6. The method according to any one of claims 1-5, wherein the precursor fiber protein is a procollagen or a derivative or fragment thereof.
7. Verfahren nach einem der Ansprüche 1-5, wobei das Vorläufer-Faserprotein ein Tropoelastin oder ein Derivat bzw. Fragment davon ist.7. The method according to any one of claims 1-5, wherein the precursor fiber protein is a tropoelastin or a derivative or fragment thereof.
8. Verfahren nach einem der Ansprüche 1 - 6, wobei das Faserprotein ein Kollagen oder ein Derivat bzw. Fragment davon ist.8. The method according to any one of claims 1-6, wherein the fiber protein is a collagen or a derivative or fragment thereof.
9. Verfahren nach einem der Ansprüche 1-5 und 7, wobei das Faserprotein ein Elastin oder ein Derivat bzw. Fragment davon ist. 9. The method according to any one of claims 1-5 and 7, wherein the fiber protein is an elastin or a derivative or fragment thereof.
10. Verfahren nach einem der Ansprüche 1 -9, wobei das Vorläufer-Faserprotein prozessierende Protein eine Lysinoxidase ist.10. The method according to any one of claims 1-9, wherein the precursor fiber protein processing protein is a lysine oxidase.
11. Pflanzenzelle, exprimierend ein Vorläufer-Faserprotein und ein es prozessierendes Protein.11. Plant cell expressing a precursor fiber protein and a protein processing it.
12. Pflanzenzelle nach Anspruch 11, wobei die Pflanzenzelle in Form eines Vermehrungsmaterials vorliegt.12. Plant cell according to claim 11, wherein the plant cell is in the form of a propagation material.
13. Pflanzenzelle nach Anspruch 11, wobei die Pflanzenzelle in Form einer Pflanze vorliegt.13. Plant cell according to claim 11, wherein the plant cell is in the form of a plant.
14. Pflanzenzelle, exprimierend ein Vorläufer-Faserprotein prozessierendes Protein.14. Plant cell expressing a precursor fiber protein processing protein.
15. Pflanzenzelle nach Anspruch 14, wobei die Pflanzenzelle in Form eines Vermehrungsmaterials vorliegt.15. Plant cell according to claim 14, wherein the plant cell is in the form of a propagation material.
16. Pflanzenzelle nach Anspruch 14, wobei die Pflanzenzelle in Form einer Pflanze vorliegt.16. A plant cell according to claim 14, wherein the plant cell is in the form of a plant.
17. Verwendung der Pflanzenzelle nach einem der Ansprüche 11 - 16 zur Herstellung eines Faserproteins.17. Use of the plant cell according to one of claims 11 - 16 for the production of a fiber protein.
18. Faserprotein, hergestellt nach dem Verfahren nach einem der Ansprüche 1 - 10.18. fiber protein, produced by the method according to any one of claims 1-10.
19. Faserprotein nach Anspruch 18, wobei das Faserprotein ein Kollagen oder ein Derviat bzw. Fragment davon ist.19. The fiber protein of claim 18, wherein the fiber protein is a collagen or a derivative or fragment thereof.
20. Faserprotein nach Anspruch 18, wobei das Faserprotein ein Elastin oder ein Derivat bzw. Fragment davon ist. 20. The fiber protein of claim 18, wherein the fiber protein is an elastin or a derivative or fragment thereof.
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WO2023194333A1 (en) 2022-04-04 2023-10-12 Swiftpharma Bv Recombinant spider silk-reinforced collagen proteins produced in plants and the use thereof

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997004123A1 (en) * 1995-07-19 1997-02-06 Gel Tech Group Inc. Collagen compound production in plants
US5928922A (en) * 1996-04-10 1999-07-27 Academy Of Finland α2 subunit of prolyl-4-hydroxylase, nucleic acid sequences encoding such subunit and methods for producing the same
AU3056697A (en) * 1996-04-12 1997-11-07 Academy Of Finland Synthesis of human procollagens and collagens in recombinant dna systems
AUPO156596A0 (en) * 1996-08-09 1996-09-05 University Of Sydney, The Synthetic polynucleotides
CA2263730A1 (en) * 1996-08-15 1998-02-19 Agrivax Incorporated Delivery of tolerogenic antigens via edible plants or plant-derived products
FR2757874B1 (en) * 1996-12-17 2003-04-25 Biocem RECOMBINANT COLLAGENS AND DERIVED PROTEINS PRODUCED BY PLANTS, THEIR METHODS OF OBTAINING AND THEIR USES

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0008142A3 *

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JP2002523020A (en) 2002-07-30
DE19834909A1 (en) 2000-02-17
WO2000008142A2 (en) 2000-02-17
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CA2336064A1 (en) 2000-02-17

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