SK4152002A3 - Method for production of proteinaceous substances - Google Patents

Abstract

The invention relates to a new method for production of heterologous proteinaceous substances in plant material. In the preferred method selected complete moss plants are cultivated and the desired target substances obtained from the culture medium essentially without disturbing the produced tissues and cells. The method allows a cost effective production of all manner of heterologous proteins in their respective active form under standardisable conditions.

Description

Technical field.

The present invention relates generally to the field of the production of proteinaceous substances in plant material. In particular, the invention relates to a novel process for the production of desired proteinaceous substances in mosses.

BACKGROUND OF THE INVENTION

The use of biotechnological methods for production purposes is an important possibility for mankind to produce substances that cannot be produced economically by other methods as long as they can be produced - for example, by chemical synthesis, and which are available as raw materials from insufficient quantities of natural resources. Although over 10,000 secondary plant metabolites are known, only a few of these compounds are produced on an industrial scale using plant cell cultures. These substances are predominantly pharmaceutically active secondary metabolites. Examples to be mentioned are a) berberine (4000I scale production) which has bacteriostatic and fungicidal effects (Fujita, Y. and Tabata, M. in: Plant tissue and cell culture, Plant Science, 3, 169 Green, CE, et al., (Ed.), AR Liss Inc., New York, 1987), b) shikonin (on a 750 I scale) having antibiotic and anti-inflammatory effects (Tabata, M. and Fujita, Y , in: Biotechnology in Science Science, 207-218, Day, P., et al., (Ed.), Academic Press, Orlando, 1985), and c) paclitaxel (75000 I) known more commonly as taxol; which has antitumor effects (Jaziri, M., et al., Taxus sp. Cell, Tissue and Organ Cultures and Alternative Sources for Taxoids Production: a Literature Survey, Plant Cel Tiss. Org. Cult., 46, 59-75, 1996) ).

Another important biotechnological method in which plant cell cultures are utilized is the biotransformation of digitoxin to digoxin - a drug for the heart and bloodstream. This stereospecific hydroxylation reaction is successfully performed in a bioreactor with Digitalis lanata cultures (Reinhard, E. and Kreis, W., Cultivation of Pflanziichen Zellen im Bioreactor); Bio. Engin., 5, 135-136, 1989 ) with high yields. A prior and extensive search of the use of plant cell cultures in biotechnology can be found in Muhlbach, H.-P.,

Use of Cell Cultures in Biotechnology, Biotechnol. Annu. Rev., 4, 113-176, 1998).

The development of genetic transformation methods in higher plants in the early 1990s made it possible to significantly increase the productivity of specific secondary components by plants by transforming genes with specific key enzymes corresponding to metabolic pathways. Not only transgenic intact plants were used, but also plant cell cultures. Examples include changes in bacterial lysine decarboxylase expression in transgenic Nicotiana tabacum root fiber cultures that increased the yield of biogenic cadaverine and anabazine amines by up to 14 fold (Berlin, J., et al., Genetic modification of plan secondary metabolism. overexpression of amino acid decarboxylases, in: Advances in Plant Biology, Studies in Plant Science, 4, 57-81, Ryu, DDY and Furasaki, S. (Ed.), Elsevier, Amsterdam, 1994).

The possibility of transfecting DNA into plants not only opened up quantitative and qualitative changes in plant components, but plants and plant cell cultures have also become more interesting for the production of heterologous proteins (Moffat, AS, High-Tech Plant Promise and Bumber Crop of New Products, Science, 256, 770). 771, 1992) and two principally different approaches were chosen.

One approach is to produce heterologous proteins in transgenic intact plants. In addition to the production of antibodies in transgenic tobacco plants (Ma, JK-C., Et al., Generation and assembly of secretory antibodies in plants, Science, 268, 716 719, 1995), the expression and regulated processing of human serumalbumin as in transgenic tobacco has been described. plants and transgenic potato plants (Sijmons, PC, et al., Production of properly processed human serum albumin in transgenic plants, Bio / Techn., 8, 217-221, 1990). Human epidermal growth factor (hEGF) has also been expressed in transgenic tobacco plants (Salmanian, A.-H., et al., Synthesis and expression of the gene for human epidermal growth factor in transgenic potato plants, Biotechnol. Lett., 18, 1095 (1098, 1996). However, other plants were also used. Leuencephaline has been successfully produced using Arabidopsis thaliana and Brassica napus (Krebbers, E. and Vandekerckhove, J., Production of peptides in plant seeds, Tibtech, 8, 1-3, 1990). Furthermore, transgenic plants of Vigna unguiculata (Dalsgaard, K., et al., Plant-derived vaccine protects target animals against a viral disease, Nat. Biotech., 15, 248-252) have been used to express chimeric viral particles that act as vaccines. , 1997).

The principal disadvantage of using intact plants, such as those mentioned above as examples, is the need for their growth, which is time-consuming and costly and requires a large cultivation area for production on an industrial scale. Moreover, the isolation of the desired target substances from intact plants usually requires complex manufacturing processes, especially if the composition and quality of the product must meet high requirements, as is the case for substances to be used for pharmaceutical or food purposes.

A second approach is to use transgenic tobacco cell cultures to produce antibodies. For example, antibody expression and secretion into the medium are described (Magnuson, NS, et al., Enhanced recovery of secreted mammalian protein from suspension culture of genetically modified tobacco cell, Prot. Expr. Pur., 7, 220-228, 1996) . Furthermore, since purification of heterologous proteins from cells is difficult, the secretion of the target protein into the medium provides a significant improvement. The production of recombinant pharmaceutically acceptable proteins in cell cultures is also of interest from a safety point of view since transgenic plant cells can grow exclusively in bioreactors and do not have to be deleted. Necessary culture mass has been made possible in large scale development of bioreactors for plant heterotropic cell cultures (e.g., Shuler, ML, et al., Bioreactor engineering and the technology of tap biodiversity: The Time of Taxol., Ann. NY Acad. Sci.). 1994, 745, 455-461).

The basic disadvantages of this second approach, where plant culture suspensions are used, are low growth rates, relatively slow secondary metabolite formation, inhibition of product formation at high cell densities, resulting in low bulk productivity, formation of aggregates and cell wall components, and increased cell sensitivity to shear stress. It should also be appreciated that when using heterotropic cell cultures, complex media must be equipped with multiple components, many of which are expensive. The most serious drawback that must be mentioned, however, is the occurrence of somaclonal changes in plant cell cultures in vitro, which entail quantitative and qualitative changes in the production of heterologous proteins (see, for example, Jones, MGK and Lindsey, K., Plant Biotechnology, nec). (Molecular Biology and Biotechnology; Walker, JM and Gingold, EW (Eds.), 2nd ed., Royal Soc. Of Chem., Burlington House, London, 1988). The heterogeneity of the products produced and their functional properties cannot be accepted, especially in the

-4in connection with the manufacture of pharmaceuticals and other substances required, whose official approval requires the provision of reliable quality and a standardized manufacturing process.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a process for the normalized production of heterologous protein substances in plant materials, which inevitably eliminates not only the above-described disadvantages of using intact plants, but also the disadvantages of using cell culture systems.

This object is achieved according to the invention by introducing a novel method of producing heterologous protein substances in plant material, in which fully differentiated moss plants are allowed to grow under normalized conditions and the protein substances formed are recovered from the culture medium without disturbing the production tissues or cells.

As used herein, the term "proteinaceous matter" refers to peptides, polypeptides and proteins, as well as fragments thereof, which are particularly suitable for diagnostic, clinical, pharmaceutical and food purposes. It also includes molecules having a peptide bond and whose information is transmitted through plant material.

In a preferred embodiment of the present invention, the desired heterologous protein substances are released into the culture medium in a biologically active form.

The term "biologically active" as used in the present description means that the target substances provided with this attribute have the functional properties required or required for the corresponding purpose. For example, if it is desired to produce antibodies, the produced protein or functional fragment thereof is biologically active as long as it is capable of inducing putative specific antigen binding. It is clear to the skilled artisan that not always a full protein is required for this purpose, but that epitopes or low molecular weight structures can be screened to provide the desired biological activity or function. For example, an enzyme is biologically active if it is able to convert its target substrate.

In a further preferred embodiment of the invention, the plant material is allowed to grow in the form of intact moss plants in a culture medium which is absolutely free of sugars, vitamins and phytohormones or functional fragments thereof.

The method of the invention allows the growth of intact and fully differentiated plants under photoautotropic conditions that can be normalized, i. without requiring the addition of sugars, vitamins and phytohormones and the like, as was the case with the previous system utilizing heterotropic cell culture suspensions. Since a cheap and simple culture medium is used, the various steps of obtaining and purifying the desired target substances in a significant amount are achieved.

The plant material to be used according to the method of the present invention is preferably an intact moss plant selected from the group of mosses including Physcomitrella, Funaria, Sphagnum and Ceratodon, and particularly preferred Marchantia and Sphaerocarpos. The process according to the invention is preferably carried out using Physcomitrella patens moss.

In another preferred solution, the nucleic acid backbone is used to transform coding not only of the desired protein species but also to convert peptides to secrete the substance from the host cell into the culture medium. As will be appreciated by those skilled in the art, any autologous and heterologous nucleic acid sequence that can be used to create an expression cassette for transforming the working tissue can be used according to the invention. In particular, the use of signal peptides is preferred for the transport of endoplasmic reticulum or cells.

The work carried out in the present invention confirms that the above-described problem of somaclonal variation is contemplated. in cell cultures, it does not exist in photoautotrophic liquid moss cultures. The mosses used according to the invention have, in addition to other systems, a clear sequence of well-defined differentiation stages (chloronema, caulonema, eye, gammophores) which can be influenced by the addition of hormones (indole-3-acetic acid induces caulonema, isopentenyladenine induces Ashton, NW, et al., Analysis of gametophytic development in the moss, Physcomitrella patens, using auxin and cytokinine resistant mutants, Planta, 144, 427-435, 1979. Thus, directed differentiation-specific expression in culture bioreactors is possible.

The heterologous proteins and the synchronously separated, pure and hence homogeneous chloroneme culture according to the invention are particularly suitable for their controllable and uniform protein production in the bioreactor and for their suitability for use in hormone-dependent or differential-specific promoters.

In addition to such expression systems, it is also possible according to the invention to use an inducible promoter system, in particular for the production of proteins with a short half-life or cytotoxic, where the β-promoter of Agrobacterium tumefaciens is preferably used.

Cultivation of mosses designed according to the invention for the production of heterologous proteins under economic conditions can be carried out, for example, using Physcomitrelly in volumes ranging from 20 ml to 6 L to 10 L and above in mixed cultures or aerated glass tanks (see, for example, Reski, R., Zell). - und Molecularbioligische Untersuchungen der cytokinin-induzierbaren

Gewebedifferenzierung und Chloroplastentilung bei Physcomitrella patens (Hedw.) B.

S.G., [Cell and molecular biology studies of cytokinin-inducible tissue differentiation and chloroplast division in Physcomitrella patens (Hedw.) B.S. J. doctoral dissertation, University of Hamburg, 1990). Because it is a culture of differentiated photoautotropic plants, it does not require medium to supply plant hormones, vitamins or sugars. Compared to the requirements for complex media, such as animal cell cultures, the cost is lower to a hundred times. According to the invention, it appears that the yield of the biologically active heterologous protein can be increased in the culture medium by up to 35 times in the presence of PVP, and therefore, according to the invention, it is preferred to use PVP in the culture medium.

Detailed information on the cultivation of other mosses in bioreactors which are suitable according to the invention, such as Leptobryum pyriforme and Sphagnum magellanicum, is described in previous works (see, for example, Wilbert, E., concerning moss cultures with particular reference to arachidonic acid metabolism, doctoral dissertation, University of Mainz, 1991; Rudolph, H. and Rasmussen, S., Studies on secondary metabolism of Sphagnum cultivated in bioreactors, Crypt. Bot., 3, 67- 73, 1992). For the purposes of the present invention, it is particularly preferred to use Physcomitrelly, especially since all the usual molecular

-Ί biological technologies (for research see Reski, R., Development, genetics and molecular biology of mosses, Bot. Acta, 111.1 - 15.1998).

Appropriate transformation systems have been developed for the biotechnological use of Physcomitrelly to produce heterologous proteins. Successful transformations were performed, for example, by direct transfer of DNA into photonema tissue using particle loading. PEG-mediated DNA transfer to moss protoplasts was also successful. This transformation method has been repeatedly described in Physcomitrelle and has led to both transient and stable transformants (see, for example, Reutter, K. and Reski, R., Production of heterologous protein in bioreactor cultures of fully differentiated moss plants, Pl. Tissue culture, Biotech 1996, 2, 142-147).

Although the present invention is principally suitable for the production of any proteinaceous substance, the production of the corresponding pharmaceutical protein will be shown herein with particular reference to human vascular endothelial growth (VEGF).

VEGF was first isolated by Ferrar, N. and Henzel, WJ (Pituitary follicular cell secrete and novel heparin-binding growth factor specific for vascular endothelial cell, Biochem. Biophys. Res. Commun., 161, 851-858, 1989) and characterized as a regulatory factor for directed angiogenesis and endothelial cell division under normal physiological conditions (Ferrara, N., et al., 1991, 1991, Cell Cell Biochem. 47: 211-218, 1991). The authors also report that this growth factor acts highly specifically on vascular endothelial cells and is inactive with respect to other cell types. VEGF is a disulfide bonded homodimeric glycoprotein. Four forms of human VEGF are known. The four isomeric forms are amino acids 121, 165, 189 and 206 in length and are formed by alternatively linked VEGF RNKs. VEGF206 was detected only in fetal liver cDNK, whereas VEGF ₁₂ i, VEGF ₁₆₅ and VEGF ₁₈₉ were detected in a large number of tumor cells and tumor tissues. All isomeric forms of VEGF had secretion sequences, but only the two smallest forms are effectively secreted (see, for example, Martiny-Baron, G. and Marme, D., VEGF-mediated tumor angiogenesis: A new target for cancer therapy, Curr. Opin. Biotechnol., 6, 675-680, 1995).

A substantial amount of VEGF has been and is still required both for the development and improvement of current requirements in tumor therapy and for characterization of VEGF. At the early stages of work on the present invention, all that has been described up to this point was the production of recombinant VEGF in insect cells by expression of the bacilovirus system.

(E.g., Fiebich, BL, et al., Synthesis and Assembly of Functionally Active Human Vascular Endothelial Growth Factor Homodimers in Insect Cell, Eur. J. Biochem., 211, 19-26, 1993). Followed by Saccharomyces cerevisiae (Kondo, S., et al., The shortest isoform of human vascular endothelial growth factor / vascular permeability factor (VEGF / VPF ₁₃₁ ) produced by Saccharomyces cerevisiae promotes both angiogenesis and vascular permeability, Biochim. Biophys. Acta, 1243, 195-202, 1995), yeast Pichia pastoris (Mohanraj, D., et al., Expression of biologically active vascular endothelial growth factor in Yeast, Growth factors, 12, 17-27, 1995) and Escherichia coli (Siemeister, G., et al., Expression of biologically active isoforms of tumor angiogenesis factor VEGF (Escherichia coli, Biochem. Biophys. Res. Commun., 222, 249-255, 1996) as other production organisms. Biologically active VEGF was produced by all of these recombination systems. However, the E. coli expression system is complicated in terms of protein purification and reconstitution as it closes into the body cavities.

DETAILED DESCRIPTION OF THE INVENTION

summary

Introduction of controllable mass cultures of Physcomitrella patens (Reuter and Reski, loc. Cit.) And methods of transfecting DNA into the moss of Physcomitralla patens (Reutter, K., Expression heterologer Gene in Physcomitrella patens (Hedw.) BSG [Expression of heterologous genes after Physcomitrella patens (Hedw. .) BSG], a doctoral dissertation at the University of Hamburg, 1994) created the basic prerequisite for the biotechnological use of this plant.

Initial work has shown long-term stability of integration using Physcomiterella transgenic lines originating from Reuterra (loc. Cit., 1994). Expression of the heterologous npt II and gus genes used as an example for this purpose was still detectable after four years. The physcometerella bioreactor culture was optimized. It was developed by the stirrer, the crushed protonema thus ensured the required homogeneity of the culture of the stable rotational speed of 300 to 500 liter-minute ^'first Standardized sampling was thus enabled. At the same time, the air supplied was more evenly distributed in the liquid culture. Under these conditions, it has been shown that the development of biomass and protein proceeds without external pH regulation in the same way as

-9 pH regulation; surprisingly, pH control is therefore not necessary. On a pilot scale, 500 mg of biomass dry matter or 22 mg of total protein per liter was produced weekly. This means a five-fold increase in biomass production in culture in a 5 L glass flask. Reducing the salt concentration in Knop's medium to one-tenth resulted in similar values and thus reduced costs.

Addition of 5 mM ammonium tartrate accelerated biomass development by shortening the lag phase. The addition of ammonium tartrate produced concurrent cultures that virtually consisted exclusively of chloroneme cells. Using flow cytometry, it was shown that virtually one hundred percent of the cells of these cultures were in the G2 / M cell cycle phase. This result was confirmed by further physiological studies with auxin and studies with differentiation-specific mutants cal 112 and cal 113 and it was found that caulonema cells are in G1 / G0 phase most of the time, while chloronema cells are predominantly in G2 / M phase.

The promoter was investigated for possible inducibility into moss using an agrobacterial 1'-promoter. The β-glucuronidase (gus) gene was used as a marker gene. Expression of the gus gene was observed in transiently transformed moss protoplasts (transformation rate = 3.10 ^-4 ) after induction with 5 μΜ by indole-3-acetic acid. No expression was observed in the control samples.

Gene "spliced" form of human vascular endothelial growth factor (VEGF, and ₂₎ amino acid 121 is converted to a transformation rate of 0.5.10 Physcomitrelly ^'5 and 3,3.10' ^6th To date, the gene has been cloned from the constitutive 35S promoter and into the pRT99 transformation vector that is suitable for plants. In the second approach, the sequence encoding the corresponding human transit peptide ER was additionally cloned. Southern analysis confirmed the integration of heterologous DNA and the described type of integration of the obtained stable transformants. Other analysis in these transformants confirmed the existence of npt II and two VEGF transcripts. Expression of VEGF ₁₂ even in moss cells was demonstrated by indirect immunofluorescence. The protein was undoubtedly localized in the cells using a confocal laser scanning microscope. These studies revealed on transformants without transit peptides that the protein is localized mainly in the cytoplasm. Transformants that additionally contained the ER transit peptide could be found to have a protein in the nucleus region and in the apical regions of the apical cells of the very high ER region. Biological activity of heterologous proteins

The 10 products produced according to the invention were verified by performing an ELISA and two functional VEGF protein assays obtained from the culture medium.

Materials and methods

Analytical grade chemicals were obtained from Fluka (Neu-Ulm), Merck (Darmstadt), Roth (Karlsruhe), Serva (Heidelberg) and Sigma (Deisenhofen) unless otherwise indicated below.

The solutions were prepared from purified pyrogen-free water, hereinafter referred to as H ₂ O, obtained from a Milli-Q water purification system (Milipore, Eschborn).

Restriction endonucleases, DNA-modifying enzymes, and molecular biology kits were obtained from AGS (Heidelberg), Amersham (Braunschweig), Applied Biosystems (Weiterstadt), Biometra (Gottingen), Boehringer Mannheim GmbH (Mannheim), Genomed (Bad Oeynhausen), New England. Biolabs (Schwalbach / Taunus); Novagen (Madison, Wisconsin, USA); Pharmacia (Freiburg); Qiagen (Hilden); and Stratagene (Heidelberg). Unless otherwise stated, they were used in accordance with the manufacturer's instructions.

Vectors and structures

Plasmid pCYTEXP-VEGF ₁₂₁ is a derivative of pCYTEXPI (Belev, TN, et al., A fully modular vector system for the optimization of gene expression in Escherichia coli, Plasmid, 26, 147-150, 1991), in which human VEGF cDNK is integrated. ₁₂ for expression into E. coli. The VEGF121 cDNK is excised from pCYTEXP-VEGF ₁₂₁ using the restriction endonuclease Nde I and Sal I, purified, blunt and cloned into the cleavage Sr I position of pRT101 (Topfer, R., et al., A set of expression vectors for transcriptional and translational) fusions, Nucleic Acids Res., 15, 5890, 1987) between the 35S promoter and the CaMV polyadenylation sequence. Using the Hin dIII, the cassette thus obtained is again excised and cloned into the restriction cleavage position of the Hin dIII transformation vector pRT99. pRT99 contains not only multiple cloning sites, but also a neomycin phosphotransferase gene under the control of the 35S promoter and the corresponding CaMV polyadenylation sequence (Topfer, R., et al., Versatile Cloning Vectors for Transient Gene Expression and Direct Gene Transfer in Cell Cell, Nucleic Acids Res. 16, 8725 (1988). This gene confers resistance to the antibiotic G418 in stable transformed plants. The plasmids were replicated on the strain

11 DH5 (Escherichia coli (Sambrook, J., et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989)).

Using the Bam HI and Big II restriction enzyme, the cDNK is excised from the vector pVE121, which was originally prepared for expression of VEGF ₁₂₁ in insect cells, and which, in addition to the VEGF ₁₂₁ sequence, contains DNA encoding a natural transit peptide that mediates secretion into animal cells. media via endoplasmic reticulum (Fiebich, et al., Synthesis and assembly of functionally active human vascular endothelial growth factor homodimers in insect cell, Eur. J. Biochem., 211, 19-26, 1993) and cloned into pRT99 using RT101 and verified.

Plasmid pNA201 is a derivative of the binary vector pBI101 (Jefferson, A.R., et al., Assaying chimeric genes in plants: The GUS gene fusion system, Plant Mol. Rep., 5, 387-405, 1987). It contains the nptII gene in the nopaline synthase promoter as a plant marker. The gus gene, which is also present, is regulated by the β-promoter from Agrobacterium tumefaciens. pNA201 is suitable for direct transformation of Physcomitrella patens.

antibody

VEGF protein is used! two different antibodies. The first antibody is a rabbit antiVEGF antibody and is directed against a synthetic peptide that corresponds to amino acids 1 to 20 of VEGF of human origin (Fiebich, et al., Loc. Cit., 1993). The second antibody is a monoclonal mouse antibody directed against human VEGF ₁₂₁ protein (R (D Systems, Wiesbaden)).

Plant material

Wild strains of moss Physcomitrella patens (Hedw.) B.S.G, originating from the genetic group of the Department of General Botany of the University of Hamburg, are used. The original strain 16/14 was collected by H. L. K. Whitehouse in Gransden Wood, Huntingngonshire (England) and further cultured from spores.

The wild strain grows either in liquid culture with Knop's medium (Reski, R. and Abel, WO, Induction of budding on chloronates and caulonemes of the moss, Physcomitrella patens, using isopentenyladenine, Planta, 165, 354-358, 1985) or on solid Knob medium with 1% Oxoid agar (Unipath, Basingstroke, England). Liquid cultures were treated as described by Reski (loc. Cit., 1990).

Bioreactor cultivation

For bulk cultivation, the material was introduced into a 7 L bioreactor through a round bottom glass flask equipped with a double jacket (Applikon Biotek, Knullwald). An air cooler, an aeration tube, a pH electrode (Conducta, Gerlingen), a temperature sensor, a stirrer, a sampling tube, and acid, base and medium inlets were introduced into the bioreactor system from above through openings in the culture chamber. The cultures were left at 25 ° C and this temperature was controlled by a suitably adjusted water bath attached to the double jacket. In experiments conducted at controlled pH, the pH was maintained at 5.8 using a titration unit. Temperature measurement and pH control was performed with a Biocontroller ADI 1030 (Applikon Biotek, Knullwald). The stirrer speed can be varied with the ADI 1012 motor controller (Applikon Biotek, Kndllwald). The culture medium is aerated with a constant air pressure of 1 bar through a porous injection element. In order to ensure the sterility of the culture vessel, all inlet and outlet pipes are equipped with filtering firing units (Midisart, 0.2 (m; Sartorius, Gottingen).) Cultivation in the bioreactor is performed in a controlled ambient lighting area (white light; Osram L 40 W) 100 (mol.s ^-1 .m ² ) The cultures are inoculated in sterile conditions with 0.5 to 1 g of undried plant material per liter of bioreactor culture The sampling tube is placed at the same level as the stirrer, thus ensuring a uniform mixer. Small samples (<100 ml) are taken through the Luer lock connection with a syringe, while bulk samples are taken with, for example, a peristaltic pump type 302 / 3A equipped with a 501 Rl head (Sartorius, Gottingen).

Wild-type chloronem cultures are developed by supplying Knop medium with 5 mM ammonium tartrate.

The yield of the biologically active heterologous protein that is secreted can be significantly increased in the presence of a polyvinylpyrrolidone (PVP) stabilizer in the culture medium.

The differentiation of caulonema that occurs during the development of photonema can be induced and increased by exogenous addition of physiological amounts of auxin of a suitable concentration such as 5i {mol / ^l indole-3-acetic acid (IAA).

In the operation of liquid culture of the transformants under pressure to Knop medium added 50 mg · l ¹ of the antibiotic G418 (Calbiochem, Bad Soden). Finally, the cultures are removed by filtration through sterile 100 µm sieves (Wilson Sieves, Nottingham, England)

13 every ten days just before crumbling and transferred to an Erlenmeyer flask filled with the selected medium.

For nutritional experiments, Knop's medium is diluted 1:10 with water.

transformation

The transformation method chosen is direct PEG-mediated DNA transfer into protoplasts as described by Reutter and Reski (loc. Cit., 1996). For each transformation, 50 [mu] g of plasmid DNA were used per ^{3 * 10 5} protoplasts. Protoplast regeneration and selection of stable transformants are made as described by Reuter and Reski (loc. Cit., 1996) unless otherwise noted.

Indirect immunofluorescence

Offset solution: MSB: 100 mM PIPES: 5-10 mM EGTA, 5 mM MgSO ₄₎ pH 6.8

F-MSB: MSB + 5% DMSO

E-MSB: MSB + 5% DMSO + 5% Nonid

W- MSB: MSB diluted H ₂ O 1: 2 (flushing buffer)

Enzyme solution: 1% cellulase, 1% pectinase, 2% driselase in MSB, pH 5.6 (all from Sigma, Deisenhofen).

Moss photonematies are fixed in 1.25% glutaraldehyde in F-MSB (v / v) by incubation not longer than 10 minutes and briefly rinsed in W-MSB. The photonematics are then incubated with 2% paraformaldehyde in MSB (v / v) for 40 minutes and rinsed 3 times with W-MSB (1 rinse; 2 washes for 5 minutes).

Free aldehydes that cannot be removed by flushing are reduced by adding MSB and solid boron hydride to the tip of the knife at an incubation time of 10 minutes. Boron hydride is removed by washing three times with W-MSB.

In the next step, the cell walls are permeated by further addition of enzyme solution for 10 minutes. The enzymatic reaction is stopped by changing the pH (MSB, pH 6.8). The mixture was rinsed 3 times with W-MSB.

Chlorophylls are extracted by incubation with detergent solution for 120 minutes. Then the solution is removed three times and extracted by incubation with detergent solution for 120 minutes. The solution was then removed by washing three times with W-MSB.

After this preparation, moss photonematics can be incubated with primary antibody (anti-VEGF; 1:50 dilution). This is done at 37 ° C for 45 minutes. After three washes with W - MSB, labeled secondary antibody (rabbit or mouse; 1:30 dilution; fluorescein isothiocyanate (FITC) labeled (Molecular Probes, Leiden, The Netherlands)) is added for 45 minutes at 37 ° C. In addition to the above three rinsing steps, the mixture is rinsed once more with W-MSB + 0.1% Triton. The photonematics are then placed in W-MSB and stored at 4 ° C for at least overnight.

The material is evaluated using a confocal laser scanning microscope (CLSM) type TCS 4D (Leica Lasertechnik, Heidelberg) and Scanware 5.0 software (Leica Lasertechnik, Heidelberg).

For analysis of CLSM samples, these are placed on a slide in mounting solution (Dabco, Sigma, Deisenhofen). The excitation of the binding of the fluorescent dye FITC to the secondary antibody is performed using an argon-crypton laser at a wavelength of 488 nm. FITC emits radiation at a wavelength of 528 nm.

ELISA Assay

The VEGF protein in the culture medium, which is produced in suitably transformed moss plants, is qualitatively determined and quantitated by conventional methods using ELISA assays and the antibodies described above. 200 µl of culture medium is used directly for ELISA.

Functional tests

The biological activity of recombinantly produced VEGF obtained from the culture medium is checked by a mitogenic assay (Miyazono, et al., 1987, Purification and properties of an endothelial cell growth factor from human platelets, J. Biol. Chem., 262, 4098-4103) and " 13 day chorioallanto-membrane angiogenic assay '(Wilting, et al., A morphological study of rabbit corneal assay, Anat. Embtyol., 183, 1167-1174, 1991). First, the culture medium is subjected to ultrafiltration, then lyophilization and subsequently resuspended in the buffer. If desired, a further purification step on a cation exchange column can be used.

Inducibility of 1 promoter

The inducibility of the β-promoter with auxin was assayed by 5 µl mol ^-1 indole-3-acetic acid (IAA). Five days after transformation, 100q aliquots of the pNA201 transformation reaction were transferred to wells of a 96-well microtiter plate (Nunc, Wiesbaden). The protoplast suspension is incubated for five hours with IAA (final concentration = 514M). Induction experiments are evaluated directly after the incubation period by a qualitative β-glucuronidase assay.

Qualitative test with β-glucuronidase

Β-glucuronidase activity is determined by a qualitative assay (Jefferson, A.R., Assaying chimeric genes in plants: The GUS gene fusion system, Plant Mol. Rep., 5, 387-405, 1987).

Substrate buffer: 50 mM K ₃ Fe (CN) ₆ mM Na ₂ HPO ₄ mM K ₄ Fe (CN) ₆ % Triton X-100 (v / v) mM NaH ₂ PO ₄ mM EDTA, pH 7.0 mg. ml ^-1 PVP (MW 10000)

Staining solution: 12.5 mg of 5-bromo-4-chloro-3-indolylglucuronic acid (Biomol, Hamburg) was dissolved in 250 µl of Ν, Ν-dimethylformamide with 50 ml of substrate buffer.

The moss photonematics and moss protoplasts in Kick or Regeneration Medium are incubated for 72 hours at 37 ° C in an equal volume of staining solution and immediately evaluated microscopically.

The results

Bioreactor culture homogeneity; sampling

Only homogeneous cultures will ensure that the sampling of cell cultures can be normalized. In prolonged cultivation, the growth of long-fiber photonems often leads to cell aggregates and thus to an inhomogeneous distribution of plant material in liquid cultures. To avoid such aggregations, photonematics crumble at certain time intervals, in a bioreactor every day following Day 10 and in mixed cultures every day 12 using high shear homogenizers. In order to maintain smooth conditions in the bioreactor with simultaneous standardized sampling and an extended cultivation period, it is recommended to adjust the turbine mixer with three mixing knives by grinding the mixing edges.

- 16 arms to transfer them to cutting arms. Constant stirring at 300-500

1.min ^-1 allows to maintain homogeneous cultures in the bioreactor.

The development of biomass (in DW [mg.l ^-1 ]) over 35 days (840 hrs) at 500 l.min ^-1 in control cultures with a turbine stirrer is the same as that in the scissor arm mixer bioreactor cultures.

The homogeneity of the culture is tested by comparing six concurrent samples. The dry matter of plant material from a sample volume of 100 ml is determined as a relative value. When using an untreated stirrer, the standard deviation increases as the biomass concentration increases. As a result of mixing with the agitator by the cutting arms, the standard deviation of the samples taken remains low. This makes it possible to conclude that a uniform homogenous culture can be obtained over 35 days with the conditioned stirrer.

1-Promoter Inducibility Study

In plasmid pNA201, the 1 promoter is located above the gus gene and acts as a regulatory element. In induction experiments with moss, the β-glucuronidase assay is known as a detection assay. Experiments with transgenic tobacco show that the 1-promoter leads to the expression of β-glucuronidase in a high auxin-containing tissue and thus it can be concluded that this promoter is auxin-dependent. Inducibility of the 1'-auxin by auxin in Physcomitrella patens is studied in transit-transformed protoplasts.

Transformation reactions are subjected to a β-glucuronidase assay with and without a 5 hour incubation with 5 gM indole-3-acetic acid (IAA). In control tests without the addition of IAA, no blue protoplasts were detected under any microscope evaluation in any reaction. In contrast, evaluated protoplasts incubated with auxin confirm the expression of the gus gene. Based on the assessment of blue protoplast transformation rate could be reached 10/3 ^'4 This is a clear sign that the D- promoter in transit transformed moss protoplasts inducible plant hormone auxin.

Generation of vectors for VEGF transformation

For the transformation of VEGF ₁₂₁ cDNK proteins without a guide sequence hereafter referred to as VEGFC and VEGF121 cDNK proteins with a guide sequence hereafter referred to herein

VEGFP to Physcomitreil it is necessary to clone the sequences between the promoter and terminator units, which is suitable for plants. The 35S promoter was selected for this purpose

- 17 CaMV and the corresponding polyadenylation signal. Suitably prepared VEGF cDNK sequences are cloned into the Sma I restriction cleavage site of the multiple cloned positions of vector pRT101.

The resulting vectors (pRT101VEGFC 3 and VEGFP 21) are sequenced with an average derived from the terminal region of the 35S promoter and verified for proper integration between the promoter and the polyadenylation position.

The resulting cassettes were excised with the restriction enzyme Hin dIII and cloned into the actual transformation vector pRT99 at the Hin dIII site (pRT99VEGFC 3 and VEGFP 21). The orientation of the cassettes with respect to the NPTII cassette can be determined by restriction with Sma I and Hinc II. If the promoter is oriented to the polyadenylation orientation of the signal, a 5250 (VEGFC) and 5380 bp (VEGFP) fragment is obtained, while the opposite orientation yields a 1100 (VEGFC) / 1230 (VEGFP) and 4150 bp (VEGFC and P) fragment. Restriction analysis yields only the 5250/5380 bp fragment, and VEGFC / P cassettes can thus be incorporated into a comparable promoter orientation to the orientation of the polyadenylation signal with the nptII gene of pRT99.

Transformation of VEGFC to Physcomitrelly

The absolute transformation rate for transforming the VEGFC structure in wild-type protoplasts after repeated transitions from Knop's medium to the selected medium is 0.5 x ^{10 -5} with sustained stability of the transformants.

Confirm integration of transformed plasmids

Successful integration into plant genomes was confirmed by Southern hybridization.

The samples are both of the neo gene, npt II, I, the second, and pRT99 fragment of Nde I / Sal I, VEGFC pCYTEXP of _VEGF-12.

The signals detected throughout the uncleaved DNA transformants confirm the successful integration of the DNA plasmids into the plant genome. Whether the whole 35S VEGFC PolyA cassette is obtained even after integration is tested with Hin dlll restriction. If the cassette remains intact on integration, this restriction enzyme excites the 1100bp fragment from the complete DNA.

The VEGFC sample hybridization template reveals a 1100 bp fragment for all transformants. Thus, the integration of the full-length VEGFC expression unit, which is predisposed for correct transcription and expression of VEGF ₁₂₁ into moss, has been demonstrated.

Demonstration of transcription of heterologous genes

The transcription of heterologous VEGFC and NPTII transformant genes is determined by a non-radioactive DIG detection system using VEGF and NPTII samples. With nucleotides 760 for transcription of VEGFC and nucleotides 1100 for transcription of NPTII, the order of transcription of the in-line transcription was determined within the limits expected for each case. As expected, none of the two heterologous transcripts were detected in the control WT.

Analysis of transformants with human transit peptide

The PEG 50-mediated DNA 50 (g pRT99P 21 plasmid DNA for a single transformation reaction produces transformants that are stably stable on the selected medium. The resulting stable transformation rate is 3.3 x ^{10 -6} .

Demonstration of integration of transformed plasmid

Integration of the above-described transformants with the transit peptide was demonstrated, as described above, by Southern hybridization using the described samples, and hybridization of the entire Hin dIII digested DNA to the VEGF sample revealed a 1230 bp fragment: evidence of completeness of the integrated 35S VEGFP PolyA cassette.

Demonstration of transcription of heterologous genes

Both NPTII and VEGFP transcription can be detected as described above.

Detection of human VEGF ₁₂₁ in transgenic moss cells using a confocal laser scanning microscope.

In this way, the test protein is labeled directly in the folded cells. The expulsion is performed under a confocal laser scanning microscope which has an improved resolution compared to a conventional light microscope.

Recombinant VEGF ₁₂₁ protein - if detectable in moss cells - must be accumulated in the cytoplasm in VEGFC transformants. In VEGFP transformants it must be detectable in the ER system as long as the transit peptide in moss functions as a signal.

Expression of human VEGF ¹²¹ in moss transgenic cells has been successfully demonstrated by indirect immunofluorescence method and computer evaluation by confocal laser scanning microscope. Furthermore, it was demonstrated that in transgenic moss VEGF ₁₂ and successfully transported into the endoplasmic reticulum in the presence of the corresponding human transit peptide.

- 19 Assay for the presence of VEGF in the culture medium

A 200 µL aliquot of culture medium in the presence of PVP is assayed by ELISA and shows that moss plants transformed with an expression cassette, including a transit peptide coding sequence, are capable of releasing VEGF into the medium. Positive results make it possible to conclude that VEGF protein is present.

The assays performed to verify the biological activity of the VEGF protein released into the culture medium give as positive results as confirm that the VEGF produced according to the invention can be obtained from the culture medium with the desired biological activity.

Claims (6)

A method for producing heterologous proteinaceous substances in a plant material, characterized in that photonical moss tissue is used as the plant material and that the proteinaceous substances obtained are obtained from the culture medium without disturbing the production tissue or cells. The method of claim 1, wherein the protein substance released into the culture medium is biologically active. Method according to claim 1 or 2, characterized in that a culture medium is used which is free of sugars, vitamins and phytohormones or functional fragments thereof. Method according to any one of claims 1 to 3, characterized in that the moss tissue is selected from the group of mosses comprising kidney. Method according to claim 4, characterized in that the moss tissue is selected from mosses of the group comprising Physcomitrella, Funaria, Sphagnum and Ceratodon. The method of claim 4, wherein the moss tissue is selected from the group of kidneys including Marchantia and Sphaerocarpos.