EP1100915A2 - Proteine de fibre et sa production - Google Patents

Proteine de fibre et sa production

Info

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
Authority
EP
European Patent Office
Prior art keywords
protein
fiber protein
plant cell
plant
precursor fiber
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Withdrawn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
EP99950481A
Other languages
German (de)
English (en)
Inventor
Klaus Düring
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.)
MPB Cologne GmbH Molecular Plant und Protein Biotechnology
Original Assignee
MPB Cologne GmbH Molecular Plant und Protein Biotechnology
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
Application filed by MPB Cologne GmbH Molecular Plant und Protein Biotechnology filed Critical MPB Cologne GmbH Molecular Plant und Protein Biotechnology
Publication of EP1100915A2 publication Critical patent/EP1100915A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

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

Abstract

L'invention concerne un procédé de production d'une protéine de fibre. Ce procédé consiste à: (a) exprimer une protéine de fibre précurseur d'une cellule végétale et (b) à incuber cette protéine de fibre précurseur avec une protéine traitant cette dernière. L'invention concerne également les cellules végétales utilisables à cet effet et les protéines de fibres obtenues par ce procédé.
EP99950481A 1998-08-03 1999-08-03 Proteine de fibre et sa production Withdrawn EP1100915A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19834909A DE19834909A1 (de) 1998-08-03 1998-08-03 Faserproteine und ihre Herstellung
DE19834909 1998-08-03
PCT/DE1999/002359 WO2000008142A2 (fr) 1998-08-03 1999-08-03 Proteine de fibre et sa production

Publications (1)

Publication Number Publication Date
EP1100915A2 true EP1100915A2 (fr) 2001-05-23

Family

ID=7876235

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99950481A Withdrawn EP1100915A2 (fr) 1998-08-03 1999-08-03 Proteine de fibre et sa production

Country Status (7)

Country Link
EP (1) EP1100915A2 (fr)
JP (1) JP2002523020A (fr)
AU (1) AU758727B2 (fr)
CA (1) CA2336064A1 (fr)
DE (1) DE19834909A1 (fr)
IL (1) IL140549A0 (fr)
WO (1) WO2000008142A2 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023194333A1 (fr) 2022-04-04 2023-10-12 Swiftpharma Bv Protéines de collagène recombinantes renforcées par de la soie d'araignée produites dans des plantes et leur utilisation

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997004123A1 (fr) * 1995-07-19 1997-02-06 Gel Tech Group Inc. Production de collagene par des plantes
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
WO1997038710A1 (fr) * 1996-04-12 1997-10-23 Fibrogen, Inc. Synthese de procollagenes et de collagenes humains dans des systemes d'adn de recombinaison
AUPO156596A0 (en) * 1996-08-09 1996-09-05 University Of Sydney, The Synthetic polynucleotides
EP0939826A2 (fr) * 1996-08-15 1999-09-08 Agrivax Incorporated Administration d'antigenes tolerogeniques via des plantes comestibles ou des produits derives de plantes
FR2757874B1 (fr) * 1996-12-17 2003-04-25 Biocem Collagenes recombinants et proteines derivees produits par les plantes, leurs procedes d'obtention et leurs utilisations

Non-Patent Citations (1)

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

Also Published As

Publication number Publication date
WO2000008142A3 (fr) 2000-06-08
WO2000008142A2 (fr) 2000-02-17
DE19834909A1 (de) 2000-02-17
CA2336064A1 (fr) 2000-02-17
AU758727B2 (en) 2003-03-27
AU6325099A (en) 2000-02-28
IL140549A0 (en) 2002-02-10
JP2002523020A (ja) 2002-07-30

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