JP2002523020A - Fibrous protein and production thereof - Google Patents

Abstract

  (57) [Summary] The present invention provides a method for producing a fibrous protein, comprising the following steps: (a) expression of a precursor fibrous protein in a plant cell; About. The invention also relates to the plant cells used for this purpose and to the fibrous proteins produced according to the method of the invention.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for producing a fibrous protein in a plant cell, a plant cell usable for this purpose, and a fibrous protein obtained by the method.

[0002] Fibrous proteins have mechanical stability, such as resilience or elasticity.
(Elastizitaet). They are formed from precursor fibrous proteins that are polymerized and cross-linked, respectively. This requires the presence of a repetitive amino acid sequence in the precursor filamentous protein and the action of the protein processing the precursor filamentous protein. Filamentous proteins are found in animal and human cells. Examples of fibrous proteins include collagen and elastin. Both are components of connective tissue, such as skin, tendons, ligaments and blood vessels. Collagen is formed by cross-linking of tropocollagen molecules, while elastin is formed by cross-linking of tropoelastin molecules.

[0003] Fibrous proteins are used for medical and cosmetic applications, respectively. For this reason, fibrous proteins are frequently isolated from animal cells. This is associated with great risk because animal diseases such as BSE can be transmitted to humans in this way.

[0004] Accordingly, it is an object of the present invention to provide a method by which a fibrous protein can be produced without said risk.

[0005] According to the invention, this is achieved by the subject-matter defined in the claims.

The present invention is capable of producing a precursor fibrous protein in a plant cell,
Next, they are based on the knowledge of the present inventors that they can be converted into the corresponding fibrous proteins by treatment with proteins that process them. In particular, the inventors have found that precursor fibrous proteins can be produced in both individual plant cells and plants. The inventor has also discovered that conversion of a precursor fibrous protein to the corresponding fibrous protein can be performed in vitro or in vivo. In the latter case, this can be done, for example, if the precursor filamentous protein is expressed in plant cells together with the protein that processes it. The present inventors have discovered using individual plant cells and plants, especially potato plants.

According to the present invention, the discovery of the inventor comprises the following steps: (a) expression of a precursor filamentous protein in plant cells, and (b) the precursor filamentous protein and a protein that processes the same. Used for the production of fibrous proteins, including incubation with

[0008] The expression "filamentous protein" includes fibrous proteins of all types and origins. It may have a two-dimensional or three-dimensional crosslinked structure. It can also be an animal or human fibrous protein. In addition, it is available in wild-type or modified form. The latter include fibrous proteins in which the amino acid sequence is modified at one or more sites relative to the wild-type sequence.
Such modifications may be additions, substitutions, deletions and / or inversions of one or more amino acids. In particular, amino acids that are suitably expressed in plant cells may be present. Alternatively, the fibrous protein may be a fusion protein, wherein the fusion partner is, for example, oleosin. This protein is then used as a plant growth material (Vermehrun
gsmaterial) to enable the localization of fibrous proteins in the oil phase. The fibrous proteins that can be utilized in the modified form have mechanical stability, for example, rebound resilience or elasticity, at least comparable to that of the wild type. Preferred fibrous proteins are collagen and elastin and any of their derivatives and fragments. For the modified forms, the above description applies correspondingly.

[0009] The term "expression of a precursor filamentous protein" includes any expression of a gene encoding a precursor filamentous protein in a plant cell, wherein the precursor filamentous protein is typically, for example, cross-linked or It can be converted to the corresponding fibrous protein by polymerization. The above description made with reference to the expression "fibrous protein" applies here correspondingly. Further, the precursor filamentous protein may be present with or without a signal peptide. The former may be, for example, a natural or foreign signal peptide that allows extracellular localization of the precursor filamentous protein. However, in the latter, the localization of the precursor filamentous protein is achieved in the cytoplasm. In addition, the precursor filamentous protein has a control peptide to allow localization of the precursor filamentous protein in certain compartments of the plant cell, such as the endoplasmic reticulum, chloroplast or vacuole. Is also good. Preferred precursor fibrous proteins are tropocollagen and tropoelastin and any of their derivatives and fragments. For expression of the gene encoding the precursor fibrous protein, an ordinary plant cell expression vector can be used. They include regulatory elements such as enhancers, promoters and termination sequences that are detected in plant cells. As an example, CaMV 35
There is an S promoter and termination sequence (Odell, JT et al., Nature 313 (1985)
), 810-812). The expression vector may include a selectable marker, for example, a neomycin or kanamycin resistance gene. In addition, expression vectors may include sequences that are convenient for their introduction into plant cells. For example, when the expression vector is introduced into a plant via Agrobacterium tumefaciens, it contains the T-DNA of a binary vector such as pSR8-30 or pSR8-35 / 1. Good (Duering, K.)
Et al., Plant Journal 3 (1993), 587-598; Porsch, P. et al., Plant Molecu
lar Biology 37 (1998), 581-585). Alternatively, the expression vector can be introduced into plant cells by a method that does not require any special sequences. Such methods include, for example, microinjection, electroporation, DNA transfer with polyethylene glycol, liposome fusion or particle bombardment.

[0010] The expression "plant cell" includes plant cells of any type and of any origin. It is a newly isolated or established individual plant cell as a cell line,
Alternatively, those present as aggregates may be indicated. The latter is, for example, a plant or a part thereof. Examples of plants include monocotyledonous plants such as corn, rice, wheat, barley and sugarcane, dicotyledonous plants such as potato, tobacco, tomato, tea, coffee, rape, especially rapeseed and cabbage, and legumes, especially Peas, kidney beans, fava beans and soybeans.

The expression “protein that processes a precursor fibrous protein” includes any protein that can convert a precursor fibrous protein to the corresponding fibrous protein. The transformation can usually be effected, for example, by crosslinking or polymerization. An example of such a protein is lysine oxidase. Also, for example, in the case of collagen, the described proteinases may be involved. Lysine oxidase and proteinase may each be present as such and as their derivatives or fragments, respectively. The above description made with respect to modified fibrous proteins applies equally to them.

The expression “incubation of precursor fibrous proteins with the proteins that process them” refers to any incubation of these proteins by which the precursor fibrous proteins can be converted into the corresponding fibrous proteins. including. The incubation may be performed, for example, in vitro. For this purpose, it is preferred to incubate the expressed precursor filamentous protein in solution with the protein that processes it. Incubation can also be performed in vivo. For this purpose, it is preferred that not only the precursor fibrillar protein, but also the protein that processes it, be expressed in plant cells. Both proteins can be expressed in different plant cells, then they are combined and the precursor filamentous protein is incubated with the protein that processes it. The precursor fibrous protein and the protein that processes it can also be expressed in the same plant cell. Thus, both proteins are automatically incubated in this plant cell. The above description made with respect to the expression of the precursor filamentous protein applies equally to the expression of the protein that processes the precursor filamentous protein.

[0013] A further subject of the invention relates to plant cells which express the precursor filamentous protein and the protein that processes it. Further, plant cells expressing only the latter among these proteins are preferred. With respect to the expressions "plant cell", "precursor fibrous protein" and "protein that processes the precursor fibrous protein", reference is made to the above description. Furthermore, the plant cells may be available in the form of a proliferative substance.

General methods can be used for producing the plant cells of the present invention. In addition to the above description, the production of a plant of the present invention expressing a precursor fibrous protein, such as tropoelastin, and a protein that processes it, such as lysine oxidase, is described as an example. . In this connection, CaMV
C encoding tropoelastin having a 35S promoter and a termination sequence
DNA is provided and binary vectors, such as pSR8-30 and pSR8
It is preferred to insert it at each of -35/1. The same can be done using cDNA encoding lysine oxidase. The resulting DNA molecule can be used to transform bacteria, for example, E. coli S17-1, suitable for transfer of the DNA molecule to Agrobacterium tumefaciens, for example, GV3101. For this purpose, E. coli S17-1 and Agrobacterium tumefaciens GV3101 are mixed with each other and incubated overnight. D
Agrobacteria incorporating NA molecules are selected by culturing in a medium containing carbenicillin. They are then subjected to the leaves of the cut potato plants, which have been scratched several times in the middle vein and incubated in the dark for 2 days. Thereafter, the Agrobacteria are removed and a growth promoter is added to the potato plants so that the shoots grow. Cut them and use them to grow new potato plants. Expression products tropoelastin and lysine oxidase and / or
Alternatively, detection of the resulting elastin is performed using specific antibodies against these proteins. Reference is made to the following examples.

Using the present invention, a fibrous protein can be produced with high purity in a plant cell, particularly in a plant. Thus, fibrous proteins are suitable for a wide variety of applications. They are found, for example, in agriculture, chemistry, cosmetics and in the manufacture of medicines.
In the latter case, for example, the use of fibrous proteins for transplantation and wound closure must be described. In particular, filamentous proteins are prominent due to the absence of animal or human viruses and pathogens, respectively. In addition, fibrous proteins can be produced in large quantities. This applies in particular when the fibrous protein is isolated from plants grown outdoors. Thus, the present invention shows that it greatly contributes to providing safe and large-scale pharmaceutical preparations.

The present invention is illustrated by the following examples.

Example 1 Production of Elastin in Potato Plants cDNA is used for human elastin (Fazio, MJ, Jour
nal of Investigative Dermatology 91 (1988), 458-464). This cDN
In A, using a PCR, an NcoI restriction site at its 5 ′ end and Xb at its 3 ′ end.
aI restriction site is provided. The obtained cDNA fragment is inserted into a vector pRT100 containing an expression cassette having a CaMV 35S promoter and a termination sequence (Tuepfer, R. et al., Nucleic Acids Research 15 (1987), 5890).
Odell, JT et al., See above). After cleavage with HindIII,
The expression cassette containing the elastin cDNA is isolated and the binary vector pSR
8-30 (Duering, K. et al .; Porsch, P. et al.
See above). The expression vector pSR 8-30 elastin is obtained.

Furthermore, cDNA for human lysine oxidase is used (Haemeleinen (Haem
aelaeinen, ER), Genomics 11 (1991), 508-516). It is processed as described above and inserted into the binary vector pSR 8-30. Expression vector pS
R 8-30 lysine oxidase is obtained.

The expression vectors pSR8-30 elastin and pSR8-30 lysine oxidase are used to transform E. coli S17-1. The transformant is mixed with Agrobacterium tumefaciens GV3101 and incubated at 27 ° C. overnight (Koncz, C., Shell, J., Molecular and Genera).
l Genetics 204 (1986), 383-396; Konkut et al., Proc. Natl. Acad. Sci. US
.A. 84 (1987), 131-135). Selection is performed with carbenicillin, and for this purpose the bla gene must be present in the expression vector. Selected clones of Agrobacterium tumefaciens are scraped several times through their middle veins, called potato plant varieties or Desirees, and incubated at 20 ° C. for 2 days in the dark but cut leaves. Thereafter, the Agrobacteria are isolated, and a growth promoter is added to the potato plant, preferably such that shoots are formed. Furthermore, non-transformed cells of a potato plant are killed by adding kanamycin to the plant medium. Cutting growing shoots, without plant growth regulators,
The roots are grown in a medium containing kanamycin. Usually, potato plants are further cultivated.

In each of the Western blot and the ELISA, analysis of the expressed tropoelastin and lysine oxidase and / or the obtained elastin is carried out with antibodies specific for the individual proteins. For this purpose, the whole protein or intracellular wash of the potato plant is isolated and used for the corresponding detection method.

It shows that tropoelastin and lysine oxidase can be expressed in plant cells, especially plants. Furthermore, it is shown that incubation of lysine oxidase with tropoelastin converts the latter to elastin, which can be isolated in pure form.

Example 2 Production of Collagen in Potato Plants Using cDNA encoding the α1 and α2 subunits of human tropocollagen (Chu, ML, et al., Journal of Biological Chemistry 260 (1985),
2315-2320; Dickson LA et al., Nucleic Acids Res. 13 (1985), 34
27-3438). Furthermore, cDNAs each encoding human lysine oxidase, human procollagen C proteinase and procollagen N proteinase derived from bovine animals are used (Haemaelaeinen, ER).
Natl. Acad. Sci USA 93 (1996), 5127-513.
0; Collige, A. et al., Proc. Natl. Acad. Sci. USA 94 (1997), 2374-2.
379).

These DNAs are treated as described in Example 1 and inserted into the pSR 8-30 vector. An expression vector, pSR 8-30 tropocollagen α1,
Obtain pSR8-30 tropocollagen α2, pSR8-30 lysine oxidase, pSR8-30C proteinase and pSR8-30N proteinase. The method is continued as described in Example 1.

It shows that tropocollagen and the proteins that process it can be expressed in plant cells, especially plants. Furthermore, it shows that high-purity collagen can be obtained.

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Claims (20)

[Claims] 1. Production of a fibrous protein, comprising: (a) expression of a precursor fibrous protein in a plant cell; and (b) incubation of the precursor fibrous protein with a protein that processes it. Method. 2. The method according to claim 1, wherein the protein to be processed is expressed in a plant cell. 3. The method according to claim 2, wherein the precursor fibrous protein and the protein that processes it are expressed in different plant cells. 4. The method according to claim 2, wherein the precursor fibrous protein and the protein that processes it are expressed in the same plant cell. 5. The method according to claim 1, wherein the plant cells are available in the form of a plant. 6. The method according to claim 1, wherein the precursor fibrous protein is procollagen or a derivative and fragment thereof, respectively. 7. The method according to claim 1, wherein the precursor fibrous protein is tropoelastin or a derivative and a fragment thereof, respectively. 8. The method according to claim 1, wherein the fibrous protein is collagen or a derivative and a fragment thereof, respectively. 9. The method according to claim 1, wherein the fibrous protein is elastin or a derivative and fragment thereof, respectively. 10. The method according to claim 1, wherein the protein that processes the precursor fibrous protein is lysine oxidase. 11. A plant cell that expresses a precursor fibrous protein and a protein that processes it. 12. The plant cell according to claim 11, wherein the plant cell is available in the form of a proliferating material (Vermehrungsmaterials). 13. The plant cell according to claim 11, wherein the plant cell is present in a plant form. 14. A plant cell that expresses a protein that processes a precursor fibrous protein. 15. The plant cell according to claim 14, wherein the plant cell is available in the form of a proliferating substance. 16. The plant cell according to claim 14, wherein the plant cell can be used in a plant form. 17. Use of the plant cell according to any one of claims 11 to 16 for producing a fibrous protein. 18. A fibrous protein produced according to the method defined in any one of claims 1 to 10. 19. The fibrous protein according to claim 18, wherein the fibrous protein is collagen or a derivative or fragment thereof, respectively. 20. The fibrous protein according to claim 18, wherein the fibrous protein is elastin or a derivative and fragment thereof, respectively.