PL224732B1 - Recombinant DNA molecule, an expression vector, recombinant protein and the use of recombinant protein molecules encoding a protein - Google Patents

Description

Description of the invention

The invention relates to a recombinant DNA molecule, an expression vector, a recombinant protein, and the use of a protein-encoding molecule. More particularly, the embodiment relates to a method of producing recombinant proteins in plants with reduced sensitivity to serine proteases. The aim of the invention is to obtain recombinant proteins representing the serine protease inhibitor CMTI I and fusions of this peptide with target proteins. This is to stabilize all proteins of the cell expressing the construct or specific protein produced as a fusion protein with a protease inhibitor.

Proteases are a group of enzymes that hydrolyze peptide bonds. They are found in viruses and all living organisms, from bacteria to mammals. They take part in such important biological processes as digestion, intracellular metabolism, protein maturation, growth and differentiation of tissues and others. Due to their properties, their activity must be strictly controlled, otherwise it can lead to serious diseases. One of the mechanisms of regulation is inhibition of their activity by protease inhibitors. A broadly represented group of inhibitors are inhibitors of serine proteases, which include, inter alia, those found in plants of the Cucurbitaceae (Otlewski and Krowarsch 1996). They are small proteins of 27-33 amino acids and have 3 disulfide bridges. Examples include the elastase inhibitor from Momordica charantia (cucumber crack), MCEI and trypsin inhibitors from the following plants: (1) Cucurbita maxima (pumpkin), CMTI I, CMTI III, CMTI IV, (2) Cucurbita pepo var Giromontia (zucchini) , CPGTI I, (3) CPTI II, Cucurbita pepo (patison), CPTI I, CPTI III, (4), Cucumis sativus (cucumber), CST Illb, CSTIIV, (5) Ecbalium elaterium (water squirt), EETI II (Wieczorek et al. 1985, Otlewski and Krowarsch 1996).

CMTI I has been very well characterized both in terms of structure, activity and mechanism of action. It is known to inhibit the activity of bovine trypsin and many other medically important serine proteases. Crystallographic and Nuclear Magnetic Resonance (NMR) studies of both the inhibitor itself and the trypsin complex allowed for the detailed study of their structure (Holak et al. 1989b, Holak et al. 1989a, Nilges et al. 1991). It is a very small protein made of 29 amino acids weighing 3.28 kDa. Three disulfide bridges stabilize and stiffen the molecule. It belongs to the group of canonical serine protease inhibitors that interact with the protease active center through the binding loop (Krowarsch et al. 2003). Non-canonical inhibitors act through the N-terminus of the protein. The peptide-binding site of Pi-P-f is located in the loop, Arg5-Ile6 for the CMTI. In the CMTI: trypsin complex, several characteristic interactions were observed, such as (1) the anti-parallel β sheet structure between the P1-P3 (Cys3) of the inhibitor and the amino acids of the 214Ser-216Gly protease, (2) van der Waales interactions between P1 and Ser 195, (3) hydrogen bonding between P1 and Gly193 and Ser195 (Krowarsch et al. 2003, Otlewski and Krowarsch 1996).

More than 15 years ago, the CMTI I gene was cloned in an Escherichia coli expression system (Bolewska et al. 1995). A mutation was introduced in the CMTI I coding sequence which resulted in an am acid change Met8> Leu. This change was necessary due to the use of cyanogen bromide protein in the purification step to cut off the sequence preceding the actual CMTI protein. The recombinant protein obtained in inclusion bodies showed biological activity in tests with bovine β-trypsin and human cathepsin G. Later studies showed that the introduced de facto mutation in the highly conserved amino acid position has a destabilizing effect on the structure and folding of the protein (Zhukov, Jaroszewski and Bierzyński 2000) .

The inhibitors have found application in plant biotechnology. In order to fight pests, plant parasites were obtained transgenic plants producing protease inhibitors that mainly inhibit the activity of digestive enzymes (Mosolov and Valueva 2008) in the digestive tract of organisms that eat them. The first reported case of obtaining transgenic plants expressing a "foreign" (originating from another organism) inhibitor gene dates back to 1987 (Hilder et al. 1987). These were transgenic plants of the Nicotiana tabacum tobacco with the introduced CpTl trypsin inhibitor gene from Chinese bean (Vigna uniguiculata). With the found production of recombinant protein in tobacco leaves at 1% TSP, it was noted that the degree of plant destruction by Heliothis virescens larvae was 50% lower compared to the control. In numerous research works, transgenic plants of tobacco, potato, rice or strawberries were obtained with increased resistance to various insects or nematodes by introducing the gene for trypsin, chymotrypsin and cystatin inhibitors

From rice or soybeans and others (Mosolov and Valueva 2008). It has also been shown that protease inhibitors can inhibit the activity of proteases secreted by fungi e.g. Fusarium solani, F. cuimorum. Increased resistance to pathogenic microorganisms was also obtained, e.g. Pseudomonas solanacearum, Botrytis, cinerea (Charity et al. 2005). However, the degree of protection in the transgenic plants obtained so far is lower than that achieved with the use of the bacterial Bt toxin gene (Bacillus thuringiensis) or with chemical pesticides (Mosolov and Valueva 2008). This may be due to the insufficient effectiveness of the inhibitors used and the ability to adapt the gastrointestinal tract of insects to their presence, e.g. by overproducing own proteases against which the inhibitors are acting or overproducing enzymes of a different class, against which these inhibitors do not work. Mechanisms are also triggered that lead to the degradation of inhibitors. In order to increase the effectiveness, attempts were made to use inhibitors derived from various organisms or modified. The introduction of the Asp86 deletion in Oc-I (rice cystatin I) expressed in the tomato hairy root culture increases the growth and development disorders of the Globodera pallida nematode (Urwin et al. 1995). Hybrid inhibitor proteins were also created, e.g. the trypsin inhibitor from CpTI Chinese bean with modified Oc-I or proteins consisting of several domains of animal and plant cystatins with equistatin (Mosolov and Valueva 2008, Urwin, McPherson and Atkinson 1998, Outchkourov et al. 2004) . Hybrids of the inhibitors with other proteins involved in the plant defense mechanism were also obtained. Transgenic tobacco with the simultaneous expression of the Bt toxin and the CpTI inhibitor showed greater resistance to insects compared to the Bt toxin alone (Mosolov and Valueva 2008). It has also been shown that the co-expression of NaPI inhibitors (trypsin and chymotrypsin inhibitor from N. alata) and StPin1A (type I inhibitor from Solanum tuberosum) protects cotton plants against Helicoverpa invasion much better than either inhibitor separately (Dunse et al. 2010).

Protease inhibitors are also used in the production of recombinant proteins in plant suspension cultures to increase the production level and stability of the recombinant protein. An example is the production of monoclonal antibodies in root suspension culture. With the co-secretion of the Bowman-Birk serine inhibitor, a significantly higher level of production of recombinant protein secreted into the medium was observed, as well as its greater stability (Komamytsky et al. 2006). Expression of the synthetic sPI-II gene having a trypsin and chymotrypsin domain from the serine II inhibitor gene of N. alata in a rice suspension culture reduced proteolytic activity to 23% compared to control (Kim et al. 2007). The positive effect of this inhibitor has also been described in the production of human granulocyte macrophage colony stimulating factor (hGM-CSF). Co-expression of both proteins resulted in a twice higher level of recombinant hGM-CSF secreted into the medium compared to the transgenic suspension culture without expression of the inhibitor (Kim et al. 2008).

Protease inhibitors, including trypsin inhibitors, and their use have been the subject of patent applications. They relate to various serine protease inhibitors and their precursors to increase plant resistance to insects and other pathogens. Predominantly, transgenic plants with the introduced protease inhibitor gene (s) are obtained. The obtained preparations of inhibitors are also used in combination with other active agents, e.g. in the fight against mites. Transgenic plants expressing the genes of the protease inhibitors SAPin2a or SAPin2b from Solanum americanum have been obtained not only for protection against insects but also as potential protective agents for heterologous proteins produced by co-expression. In order to increase the effectiveness in the fight against plant pests, a patent has also been submitted for the production of a fusion protein composed of two components with anti-pathogenic activity linked by a linker (Atkinson et al. 2005). Some patents concern medical applications of serine or cysteine protease inhibitors, e.g. the preparation of an inhibitor from the Kunitz family BTL0.10 in the treatment of diseases related to organ damage by uncontrolled proteolysis, e.g. emphysema, idiopathic pulmonary fibrosis, cystic fibrosis, rheumatoid arthritis.

Despite the conducted research and existing solutions, there is a constant need to create a solution that would enable the production of recombinant proteins representing the serine protease inhibitor, CMTI I and fusions of this peptide with target proteins, thanks to which it would be possible to stabilize all proteins of the cell expressing the construct or a specific protein, produced as a fusion protein with a protease inhibitor.

Unexpectedly, a solution was obtained regarding the CMTI itself, its expression in plants, or its fusion. The research is distinguished by the production of a fusion protein in the plant, consisting of an inhibitor and a target protein, an example of which is a cytokine, i.e. a protein of therapeutic importance.

PL 224 732 B1

The purpose of this production method is to increase the stability of the target protein both during extraction and purification as well as in the gastrointestinal tract after oral administration.

The subject of the invention is a recombinant DNA molecule, characterized in that it comprises a nucleic acid molecule encoding a protein having serine protease inhibitory activity and a nucleic acid molecule encoding a target protein, preferably selected from the group consisting of animal cytokines, wherein the sequence encoding the linker is present between the sequences encoding the two proteins. a protein, wherein all recited elements form a single open reading frame, wherein the protein having serine protease inhibitor activity is the CMTI and is defined by the sequence of SEQ ID NO: 2.

Preferably, the nucleic acid molecule is operably linked to regulatory elements comprising a promoter and a transcription terminator active in a plant cell, the regulatory element constituting the active promoter being constitutive or regulated.

Preferably, the nucleic acid molecule is operably linked to regulatory elements enabling the stable or transient expression of said nucleic acid in the plant cell.

Preferably, the nucleic acid molecule is operably linked to a DNA molecule encoding peptides capable of directing the protein to a desired compartment of the cell. Preferably, the molecule comprises a sequence coding for a factor that protects the target protein against serine proteases, especially trypsin.

Preferably, the molecule contains a sequence coding for a factor that protects the exogenous proteins against serine proteases, especially trypsin.

Another object of the invention is an expression vector, characterized in that it has at least one recombinant molecule as defined above.

Another object of the invention is a recombinant protein obtained in plants from the molecule defined above, characterized in that it has the activity of a serine protease inhibitor.

Preferably, the recombinant protein provides protection of the target protein against serine proteases, especially trypsin.

Preferably, the recombinant protein provides protection to the exogenous proteins against serine proteases.

Preferably, the recombinant protein is a "chimeric" fusion protein, wherein both the fusion elements, the serine protease inhibitor and the target protein, are linked via a protein linker, preferably having the sequence ADLQKLISEEDLHHHHHHGSG.

Preferably, the recombinant protein is a "chimeric" fusion protein, wherein both the fusion elements, the serine protease inhibitor and the target protein, are biologically active.

Another object of the invention is the use of a protein-encoding molecule as defined above to protect the molecule itself, including the target protein, and exogenously added proteins from serine proteases.

Preferably, the use of recombinant protein is used to protect exogenous proteins.

In order to better understand the invention, the figure has been used, where:

Figure 1 is a schematic representation of the CMTI expression cassette. P RbcSl and T RbcSl denote the promoter and transcription terminator gene sequences of the RUBISCO small subunit from Asteraceous chrysanthemum, respectively; PS - Hderowy peptide sequence from Anemone equistation; Inh - CMTI sequence; c, His and K - labels cmyc, 6x His and KDEL; Ascl, Pad, Ncol, BglII - restriction sites;

Figure 2 is a schematic representation of the CMTI :: Cytokine expression cassette. P RbcSl and T RbcSl denote the promoter and transcription terminator gene sequences of the RUBISCO small subunit from Asteraceous chrysanthemum, respectively; PS - Hderowy peptide sequence from Anemone equistation; Inh the CMTI sequence; GSG peptide Glycine-Serine-Glycine; cyt-hIL-2 or GM-CSF; Ascl, Pacl, Ncol, Sacl restriction sites;

figure 3 shows the detection of recombinant CMTI, CMTI :: hIL-2, CMTI :: GM-CSF proteins by Western blot. Primary anti-His antibodies were used to detect CMTIs; to detect fusion proteins of anti-hi L-2 and anti-GM-CSF antibodies, respectively. Negative control (K-), untransformed plant extract; positive control (K +), recombinant GM-CSF protein produced in E. coli; L. size marker;

figure 4 shows the inhibition of trypsin activity by a recombinant CMTI produced as a single or a fusion protein. AEBSF is a commercially available protease inhibitor used

PL 224 732 B1 as positive control; GM-CSF and hIL-2 plant extracts expressing cytokines used as negative controls;

Figure 5 shows the protection of the fusion protein from trypsin digestion. hIL-2, a protein extract derived from a plant producing recombinant hIL-2 protein; CMTI :: hIL-2, protein extract derived from a plant producing the recombinant CMTI :: hIL-2 fusion protein; L, size marker;

figure 6 shows the biological activity of the recombinant hIL-2 cytokine produced in transgenic plants as a single protein and fused with the protease inhibitor CMTI. Serial two-fold dilutions of protein extracts from transgenic plants were used to obtain the following concentrations of hIL-2 in the extract, respectively: 1 ng / ml, 0.5 ng / ml, 0.25 ng / ml and 0.125 ng / ml in dilutions (1, 2, 3 , and 4). Commercial hIL-2 from E. coli at a concentration of 25 ng / ml, 12.5 ng / ml, 6.25 ng / ml and 3.125 ng / ml was used as a positive control. The negative control was the extract of a non-transgenic plant and the medium without the addition of hIL-2. Mammalian cells of the CTLL2 line were incubated with the prepared dilutions, their proliferation was determined in the MTS experiment with OD measurement of 490 nm. The line at 0.34 means the so-called background or reading for the negative control.

Sequences

SEQ No. 1 and SEQ No. 2 show the sequence of the CMTI gene and protein with the leader peptide and labels. The mature CMTI protein sequence with cmyc and His tags is underlined;

SEQ No. 3, SEQ No. 4, SEQ No. 5 and SEQ No. 6 show the sequences of the genes and fusion proteins together with the leader peptide and labels. The sequence of the mature CMTI :: hIL2 and CMTI :: GM-CSF proteins are underlined. The linker sequence between the genes / fusion proteins is shaded.

For a better understanding, examples of embodiments according to the invention are presented below.

Examples of the application of the invention

The aim of the invention is to obtain recombinant proteins representing the serine protease inhibitor CMTI I and fusions of this peptide with target proteins. This is to stabilize all proteins of the cell expressing the construct or the specific protein produced as a fusion protein with a protease inhibitor. The presence of a serine protease inhibitor can provide protection against degradation by digestive enzymes, e.g. trypsin, during oral administration of such a protein to animals or humans as a vaccine or drug. The CMTI I peptide and the fusion proteins are preferably appropriately labeled cmyc and His (Figure 1).

Fusion proteins carrying the CMTI I domain fused to the target peptide have been shown to retain the full postulated activity of the fusion protein. Both the fusion elements, the inhibitor and the target protein, are active. The key element in maintaining the full activity of both proteins in the fusion seems to be the way of their connection through a linker, for example the linker with a length of 21 amino acids checked by us. It is known from the literature that CMTI has already been tested as a fusion protein produced in a bacterial system (Milner et al. 2007). Fusion constructs of CMTI with the potato Y virus coat protein (CP) and GUS (β-glucuronidase) were obtained. Contrary to our research, no linker was used to separate the two genes. It turned out that the CMTI strongly disrupted the activity of both fusion proteins. Moreover, the CMTI retained specific activity only when fused with CP. Contrary to this literature data, both the CMTI and the target protein retain biological activity in our system. Moreover, recombinant interleukin 2 (IL-2) fused with CMTI shows a higher specific biological activity in the CTLL-2 cell proliferation assay than IL-2 obtained in the same plant system but as a single protein. We have demonstrated the activity of recombinant CMTI to protect exogenous protein from trypsin.

Example I. Construction of an expression cassette containing a recombinant DNA molecule with a CMTI protease inhibitor gene designed for expression in plants with targeting of the protein to the ER (endoplasmic reticulum).

The CMTI-I gene encoding a trypsin inhibitor was amplified by polymerase chain reaction (PCR) on a template of a recombinant vector containing the CMTI cDNA using a specific primer pair NcoCMTI and BglCMTI. The 109 nucleotide long PCR fragment was cloned into the ImpactVector 1.3 tag after digestion with NcoI and BglII restriction enzymes. This plasmid contains the rubisco RbcSl promoter and terminator, an Anemone equistation leader peptide coding sequence, and the c-myc and 6xHis labels. The resulting expression cassette was then cloned into the pBIN PLUS binary vector into the Ascl and Pad restriction sites. The vector pBINPLUS contains the plant selectable marker, nptII, which provides kanamycin resistance. Figure 1 is a schematic representation of the expression cassette with those listed above

The most important elements. The sequences described as SEQ No. 1 and SEQ No. 2 contain the sequence of the CMTI gene and the mature protein.

P r z x l a d II. Construction of an expression cassette containing a recombinant DNA molecule with a CMTI protease inhibitor gene fused to an IL-2 or GM-CSF cytokine gene designed for expression in plants targeting the recombinant protein to the ER (endoplasmic reticulum).

The preparation of the fusion construct was carried out in two steps. In the first, in the polymerase chain reaction on a template CMTI cDNA cloned in ImpactVector 1.3, the inhibitor gene was amplified with two specific primers (restriction sites are underlined);

1. NcoCMTI: 5'-TATCCATGGCGCGTGTTTGCCCGCGTATCCTGATG-3 '

2. HisGSG: 5'-ACCGGATCCGTGATGGTG ATGGTGATGAAGATC-3 '

At the same time, the cytokine gene was amplified on the template of a recombinant plasmid containing the cDNA of the appropriate cytokine with a pair of specific primers:

1. For IL-2

a. GSGIL2: 5'-CACGGATCCGGTGCACCTACTTCAAGT-3 '

b. SacIL2: 5'-AAGAGCTCTTAGAGTTC ATCTTTAGTTAG-3 '

2. For GM-CSF

a. GSGGMCSF: 5'-CACGGATCCGGTGCACCCACCCGC-3 '

b. SacGMCSF: 5'-GGAGCTCTTAAAGCTCATCTTTTTGGAC-3 '

As a result of the performed reactions, two products of the PCR reaction were obtained, which contain a complementary fragment of several nucleotides from the 3 'end of the CMTI (CACGGATCCGGT) and the 5' end of the cytokine (ACCGGATCCGTG), which makes it possible to combine them. In the second stage of the polymerase chain reaction with primer pairs NcoCMTI, SachIL2 and NcoCMTI, SacGMCSF, a CMTI :: hIL-2 and CMTI :: GM-CSF cDNA fusion was obtained. The PCR product after digestion with NcoI and SacI was cloned into the Impact Vector 1.3 tag. The expression cassette was transferred into a binary vector as described above in Example I. Figure 2 shows a schematic of the expression cassette containing the RbcSl promo and terminator, the sequences encoding cmyc, 6xHis, KDEL, leader peptide, and the CMTI gene and cytokines. The sequence of the CMTI :: hIL-2, CMTI :: GM-CSF gene and protein fusion proteins is described by SEQ # 3, SEQ # 4, SEQ # 5 and SEQ # 6.

P r x l a d III. Transformation of tobacco plants with recombinant DNA molecules using Agrobacterium tumefaciens.

The recombinant plasmids pBIN / CMTI-KDEL and pBIN / CMTI :: hIL2-KDEL described in Examples I and II were introduced into cells of the Agrobacterium tumefaciens LBA4404 strain by electroporation. The functionality of the obtained gene constructs was checked in the transient expression system. For this purpose, Agrobacterium suspensions containing the described plasmids were injected into Nicotiana benthamiana tobacco leaves. After 72 hours, leaf samples were collected from which proteins were isolated. Then Western analysis was performed which confirmed the presence of recombinant proteins.

Nicotiana tabacum LA Burley 21 low alkaloids tobacco was selected for the stable transformation. The presence of recombinant proteins was confirmed by Western blot using anti-hIL-2, anti-mGM-CSF, anti-His or cmyc monoclonal primary antibodies. The results are shown in Figure 3. The expected size for CMTI, CMTI :: hIL-2, CMTI :: GMCSF (including KDEL, His- and cmyc-tag) is 6.14 kDa, 21.75 kDa, 21.35 kDa, respectively. . An extract from a non-transgenic plant was used as a negative control. The GM-CSF fusion proteins were N-glycosylated in plants, therefore their size is greater than that given above.

The production level of the fusion proteins was determined by ELISA and ranged from 0.48-3.38 pg / g fresh tissue (FW) for CMTI :: IL-2 and 4-12 pg / g for CMTI :: GM-CSF.

P r x l a d IV. Analysis of the activity of recombinant CMTI and CMTI :: hIL-2 proteins produced in transgenic plants

The activity of the recombinant inhibitor protein was tested in a trypsin inhibition assay using Azocoll as a substrate. CMTI and CMTI / hIL2 transgenic plant extracts obtained by straining leaf samples in a buffer containing 50 mM Tris-HCl, 2% PVP, pH 7.5. The difference in absorbance reading between the positive and negative samples reflects the activity of the inhibitor. Higher OD ₄₉₀ readings indicate the degradation of the standard protein, i.e. Azocoll, which means low inhibitor activity. The results are shown in Figure 4. They show that both the recombinant inhibitor itself and its fusion protect the standard protein from trypsin digestion. As a negative control

An extract of a transgenic plant producing the recombinant hIL-2 protein was used. The positive control was a commercial preparation of serine protease inhibitors, AEBSF [4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride].

Protection of the cytokine protein hIL-2 produced in plants in fusion with the CMTI inhibitor against trypsin digestion has also been demonstrated. The protein extract of the transgenic plant transformed with the CMTI :: hIL-2 fusion gene was incubated at 25 ° C for 30 minutes with the addition of trypsin at a weight ratio of 0.024: 1 enzyme (trypsin) to total protein, i.e. 119: 1 trypsin to recombinant protein. Samples were taken at intervals of several minutes and the presence of the recombinant fusion protein was checked by Western blot method using anti-hIL-2 antibodies. A significant decrease in the amount of recombinant protein was observed in the extract of the transgenic plant expressing hIL-2 after just 4 minutes of incubation. Almost complete loss of protein was found after 30 minutes of incubation. In contrast, in the case of the plant extract expressing the fusion protein, a slight decrease in the amount of recombinant protein was observed within 30 minutes of incubation (Fig. 5).

Example V. Analysis of the activity of recombinant cytokine proteins in fusion with the CMT1 inhibitor

The activity of the hIL-2 protein obtained in fusion with the CMTI was tested in the CTLL2 cell lineage iteration assay (murine lymphoblasts), which requires the presence of IL-2 in the medium for growth. The results are shown in Figure 6. Serial two-fold dilutions were added to the prepared cell suspensions; (1) a commercial preparation of hIL-2 (purified from Escherichia coli bacteria) to a concentration of 25 ng / ml, 12.5 ng / ml, 6.25 ng / ml and 3.125 ng / ml medium; (2) protein extracts from hiL-2 and CMTI :: hIL-2 transgenic plants, such that the concentration is 1 ng / ml, 0.5 ng / ml, 0.25 ng / ml and 0.125 ng / ml medium; (3) extracts from a non-transgenic plant as negative control. After 72 hours of incubation, the proliferation level was assessed using Promega's Cell-titer 96 Aqueous One solution assay (MIS). Comparing the results obtained for proteins produced in plants, it can be concluded that the hIL-2 protein in fusion with CTMI shows a slightly higher specific activity (i.e. per ng of protein) than the non-fused hIL-2 protein. Moreover, by comparing the OD reading levels, it can be concluded that hIL-2 of bacterial origin at a concentration of 3.125 ng ml shows just as much activity as the fusion protein at a concentration 6-fold lower, which indicates a clearly higher specific activity of recombinant IL-2 fusion and compared to IL-2 -2 of bacterial origin based on the weight of protein.

Literature

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Claims (14)

1. Recombinant DNA molecule, characterized in that it comprises a nucleic acid molecule encoding a protein having serine protease inhibitor activity and a nucleic acid molecule encoding a target protein, preferably selected from the group consisting of animal cytokines, wherein the sequence encoding the protein linker is between the sequences encoding the two proteins. wherein all specified elements form a single open reading frame, wherein the protein having serine protease inhibitor activity is CMTI I and is defined by the sequence of SEQ ID NO: 2. 2. A recombinant molecule according to claim 1 The method of claim 1, wherein the nucleic acid molecule is operably linked to regulatory elements, including a promoter and a transcription terminator, active in a plant cell, the promoter regulatory element being constitutive or regulated. 3. A recombinant molecule according to claim 1 A method as claimed in claim 1, characterized in that the nucleic acid molecule is operably linked to regulatory elements enabling the stable or transient expression of said nucleic acid in a plant cell. 4. A recombinant molecule according to claim 1 The method of claim 1, wherein the nucleic acid molecule is operably linked to a DNA molecule encoding peptides capable of targeting proteins to a desired compartment of the cell. PL 224 732 B1 5. Recombinant molecule according to claims 1 to 4, characterized in that it contains a sequence coding for a factor ensuring protection of the target protein against serine proteases, in particular trypsin. 6. Recombinant molecule according to claims 1 to 4, characterized in that it contains a sequence coding for a factor ensuring protection of the exogenous proteins against serine proteases, especially trypsin. 7. An expression vector which has at least one recombinant molecule as defined in claims 1 to 6. 8. Recombinant protein produced in plants from the molecule according to claims 1 to 6, characterized in that it has a serine protease inhibitor activity. 9. Recombinant protein resulting from the molecule according to claims 1 to 6, characterized in that it provides protection of the target protein against serine proteases, especially trypsin. 10. Recombinant protein produced in plants from the molecule according to claims 1 to 6, characterized in that it provides protection of exogenous proteins against serine proteases. 11. Recombinant plant-derived protein from the molecule according to claims 1 to 6, characterized in that it is a "chimeric" fusion protein, wherein both the fusion elements - the serine protease inhibitor and the target protein are linked by a protein linker, preferably with the sequence ADLQKLISEEDLHHHHHGSG. 12. Recombinant protein produced in plants from the molecule according to claims 1 to 6, characterized in that it is a "chimeric" fusion protein, wherein both the fusion elements of the serine protease inhibitor and the target protein are biologically active. 13. The use of a protein-encoding molecule as defined in claims 1 to 6 to protect the molecule itself, including the target protein, and exogenously added proteins from serine proteases. 14. The use of a recombinant protein made in plants from the molecule according to claims 1 to 6 for the protection of exogenous proteins.