Field of the invention

The subject of the invention are recombinant RE15mR and EJ17zipR hybrid proteins, in particular having the sequence and scheme presented below (made of domains of structural proteins: resilin, elastin, collagen or silk, with introduced cross-linking sites - additional lysines, cleavage sites for metalloproteinases and adhesion sequences of the cellular RGD), in particular of the scheme shown in Fig. 1 and Fig. 2, and the recombinant DNA sequences encoding the recombinant hybrid proteins RE15mR and EJ17zipR, in particular shown in Fig. 4 and Fig. 5. The subject of the invention also includes vectors comprising said recombinant DNA sequences of the invention and eukaryotic or prokaryotic expression systems, transformed with said recombinant DNA sequences of the invention. Finally, the subject of the invention includes use of the recombinant hybrid proteins of the invention, and the use of the recombinant DNA sequences, vectors and eukaryotic or prokaryotic expression systems of the invention. The subjects of the invention will find application in bioprinting, medicine and cell/tissue engineering.

The inventions are linked by a common inventive concept, common use and common features and properties.

Prior art

Bioprinting and biomaterials are currently intensively researched and developed. Technical solutions in these fields enable 3D printing of tissue structures, and ultimately treatment in regenerative medicine. Thus, there are examples of natural and synthetic bioinks and biomaterials in the art, such as the biopolymers disclosed in US 2022/0047706. Nevertheless, the materials used in bioinks and biomaterials still need improving their effect on cell viability, proliferation, adhesion or cytotoxicity (also in combination as a component of blends).

Aim of the invention

The aim of the invention was to propose a new, alternative recombinant hybrid protein useful in particular as a component of bioink and a biomaterial. Such a protein should be universal, safe and effective to use both in the laboratory environment, and ultimately in the patient. The aim of the invention was also to overcome the technical problems found in the state of the art, in particular by providing a protein with at least one parameter, such as the effect on cell viability, proliferation, adhesion or cytotoxicity (also in combination as a component of blends), comparable or better than in proteins currently used in the state of the art.

Summary of the invention

The subject of the invention includes a recombinant RE15mR hybrid protein composed sequentially of 3 resilin domains, 1 K+ domain, 1 MMP domain, 3 elastin domains, 1 K+ domain, 4 elastin domains, 1 K+ domain, 4 elastin domains, 1 K+ domain, 3 elastin domains, 1 MMP domain, 7 RGD domains.

Preferably, the recombinant RE15mR hybrid protein according to the invention is characterized in that it contains about 12-15% of proline and/or about 32-38% of glycine in the amino acid sequence.

Preferably, the recombinant RE15mR hybrid protein according to the invention is characterized in that its molecular weight is about 26 kDa.

Preferably, the recombinant RE15mR hybrid protein of the invention is characterized in that it has the structure shown in Fig. 1.

Preferably, the RE 15mR recombinant hybrid protein of the invention is characterized in that it has the amino acid sequence shown in Fig. 6.

Preferably, the RE15mR hybrid recombinant protein of the invention is characterized in that it is a fragment of a recombinant protein.

Preferably, the recombinant RE15mR hybrid protein of the invention is characterized in that it is at least partially methacrylated.

The subject of the inventions also includes recombinant DNA sequence encoding a recombinant RE15mR hybrid protein of the invention, or a recombinant DNA sequence comprising a region comprising a DNA sequence encoding a recombinant RE15mR hybrid protein of the invention, or a recombinant DNA sequence hybridizing to a DNA sequence encoding a recombinant RE15mR hybrid protein of the invention. Preferably, the recombinant DNA sequence of the invention is characterized in that it is at least 80% identical, more preferably at least 90% identical, most preferably homologous to the recombinant DNA sequence shown in Fig. 4.

The subject of the inventions also includes a vector comprising a recombinant DNA sequence comprising a region comprising a recombinant DNA sequence of the invention, or a recombinant sequence comprising a portion of a region comprising a recombinant DNA sequence of the invention.

Preferably, the vector according to the invention is characterized in that it is a plasmid containing a promoter derived from the T7 phage, preferably selected from pETl la-d, pET15b, pET19b, pET28a-c(+), pET21a-d(+), pET22b(+), pET23a -d(+), pEt25b(+), pET44a-c(+), pET46Ek/LIC, most preferably being pETl la.

The subject of the invention also includes eukaryotic or prokaryotic expression system transformed with a recombinant DNA sequence comprising a region comprising a recombinant DNA sequence of the invention, or a recombinant sequence comprising a portion of a region comprising a recombinant DNA sequence of the invention.

Preferably, the eukaryotic or prokaryotic expression system of the invention is characterized in that it is a CHO Chinese hamster ovary epithelial cell, or an Escherichia coli cell.

The subject of the invention includes a recombinant hybrid protein EJ17zipR, composed sequentially of 1 ZIP domain, 56 elastin domains, 9 silk fibroin domains, 7 RGD domains, 56 elastin domains, 10 silk fibroin domains, 7 RGD domains.

Preferably, the recombinant hybrid protein EJ17zipR according to the invention is characterized in that it contains about 12-17% of proline and 20-30% of glycine in the amino acid sequence.

Preferably, the recombinant hybrid protein EJ17zipR according to the invention is characterized in that its molecular weight is about 86 kDa.

Preferably, the recombinant hybrid protein EJ17zipR according to the invention is characterized in that it has the structure shown in Fig. 2.

Preferably, the recombinant EJ17zipR hybrid protein of the invention is characterized in that it has the amino acid sequence shown in Fig. 7. Preferably, the recombinant EJ17zipR hybrid protein of the invention is characterized in that it is a fragment of a recombinant protein.

Preferably, the recombinant EJ17zipR hybrid protein of the invention is characterized in that it is at least partially methacrylated.

The subject of the invention also includes a recombinant DNA sequence encoding a recombinant EJ17zipR hybrid protein of the invention or a recombinant DNA sequence comprising a region comprising a DNA sequence encoding a recombinant RE15mR hybrid protein of the invention or a recombinant DNA sequence hybridizing to a DNA sequence encoding a recombinant RE15mR hybrid protein of the invention.

Preferably, the recombinant DNA sequence of the invention is characterized in that it is at least 80% identical, more preferably at least 90% identical, most preferably homologous to the recombinant DNA sequence shown in Fig. 5.

The subject of the inventions also includes a vector comprising a recombinant DNA sequence comprising a region comprising a recombinant DNA sequence of the invention, or a recombinant sequence comprising a portion of a region comprising a recombinant DNA sequence of the invention.

Preferably, the vector according to the invention is characterized in that it is a plasmid containing a promoter derived from the T7 phage, preferably selected from pETl la-d, pET15b, pET19b, pET28a-c(+), pET21a-d(+), pET22b(+), pET23a -d(+), pEt25b(+), pET44a-c(+), pET46Ek/LIC, most preferably being pETl la.

The subject of the invention also includes eukaryotic or prokaryotic expression system transformed with a recombinant DNA sequence comprising a region comprising a recombinant DNA sequence of the invention, or a recombinant sequence comprising a portion of a region comprising a recombinant DNA sequence of the invention.

Preferably, the eukaryotic or prokaryotic expression system of the invention is characterized in that it is a CHO Chinese hamster ovary epithelial cell, or an Escherichia coli cell.

The subject of the invention includes use of the recombinant hybrid protein RE15mR according to the invention or the recombinant hybrid protein EJ17zipR according to the invention in bioprinting, as an additive or the main component of bioink. The subject of the invention includes the use of the recombinant hybrid protein RE15mR according to the invention or the recombinant hybrid protein EJ17zipR according to the invention in medicine, as an additive or the main component of a biomaterial.

The subject of the invention includes the use of a recombinant DNA sequence according to the invention, or a vector according to the invention, or an eukaryotic or prokaryotic expression system according to the invention, for the production of a recombinant RE15mR hybrid protein according to the invention.

The subject of the invention includes the use of a recombinant DNA sequence according to the invention, or a vector according to the invention, or an eukaryotic or prokaryotic expression system according to the invention, for the production of a recombinant EJ17zipR hybrid protein according to the invention.

Advantages of the invention

The invention will be used in the bioprinting process. The produced proteins will be an addition to natural bioinks (e.g. based on dECM - decellularized extracellular matrix; mixtures of single proteins / natural compounds) and synthetic ones, as an additive that improves cell survival, proliferation and adhesion. Due to the individual domains in the designed proteins according to the invention, it is also possible to use them as a basic component of the bioink (and not only as an additive, as described above).

Due to their structure (mainly collagen and elastin domains), the proteins according to the invention can be used in broadly defined regenerative medicine, which also uses other techniques (other than 3D bioprinting) for biomaterial production.

In addition to bioinks and biomaterials for the production of bionic organs and tissue systems, the proteins according to the invention can be used in regenerative medicine, e.g. as medical dressings or their components.

The proteins according to the invention will be used to create biomaterials in both methacrylated and non-methacrylated forms, which will definitely expand the possibility of their use in final products.

The proteins according to the invention have many benefits, the most important of which is the improvement of the physico-chemical and biological properties of bioprinted 3D tissue models, bionic organs and biomaterials used both in scientific research and regenerative medicine, e.g. by improving cell adhesion and growth, improving stiffness, fatigue life, elasticity or flexibility of printouts.

Brief description of the figures of drawing

The subject of the invention in the embodiment is illustrated in the attached drawing, in a way that does not limit the scope of the invention, wherein:

Fig. 1. Shows a diagram of the RE15mR recombinant hybrid protein

Fig. 2. Shows a diagram of the EJ17zipR recombinant hybrid protein

Fig. 3. Shows tables with primer sequences for cloning and sequencing genes encoding recombinant hybrid proteins

Fig. 4. Shows the coding sequence for the RE15mR recombinant hybrid protein

Fig. 5. Shows the coding sequence for the EJ17zipR recombinant hybrid protein

Fig. 6. Shows the amino acid sequence of the RE15mR recombinant hybrid protein

Fig. 7. Shows the amino acid sequence of the EJ17zipR recombinant hybrid protein

Fig. 8. Shows the map of the expression vector with the cloned gene growing the RE15mR hybrid protein.

Fig. 9. Shows the map of the expression vector with the cloned gene growing the EJ17zipR hybrid protein.

Fig. 10. Shows the composition of culture media in table form.

Fig. 11. Shows the composition of the lysis buffer in table form.

Fig. 12. Presents an image of the SDS-PAGE separation of samples taken from the subsequent stages of obtaining the recombinant RE15mR hybrid protein, where the numbers in chapter A mean: 1 - culture of E. coli BLR(DE3) before induction; 2 - E. coli BLR(DE3) culture after IPTG induction; 3 - standard of LMW protein masses; 4 - supernatant after sonication; 5 - sediment after sonication; 6 - supernatant after sonication and incubation at 90°C; 7 - pellet after sonication and incubation at 90°C; 8 - supernatant after sonication, incubation and ammonium sulphate salting-out at 0-20% saturation; 9 - sediment after sonication, incubation and ammonium sulphate salting-out at 0-20% saturation; 10 - supernatant after sonication, incubation and ammonium sulphate saltingout at 20-40% saturation; 11 - sediment after sonication, incubation and ammonium sulphate salting out at 20-40% saturation; 12 - dialysis supernatant I; 13 - dialysis sediment I, and in chapter B, the numbers mean: 1 - standard of LMW protein masses; 2 - input for the Macro-Prep High Q Media deposit; 3 - fractions not bound to the Macro- Prep High Q Media bed; 4 - fractions bound to the Macro-Prep High Q Media bed; 5 - fractions entering the Pierce High-Capacity Endotoxin Removal Resin deposit; 6 - fractions eluted from High-Capacity Endotoxin Removal Resin; 7,8,9,10 - final protein solution for lyophilization.

Fig. 13. Shows an image of the SDS-PAGE separation of samples taken from the successive stages of obtaining the recombinant EJ17zipR hybrid protein, wherein the numbers in chapter A mean: 1 - culture of E. coli BLR(DE3) before induction; 2 - culture of E. coli BLR(DE3) after induction; 3 - Broad Multi Color protein mass standard; 4 - supernatant after sonication; 5 - sediment after sonication; 6 - supernatant after sonication and incubation at 90 °C; 7 - sediment after sonication and incubation at 90 °C; 8 - supernatant after sonication, incubation and ammonium sulphate salting-out at 0-10% saturation; 9 - precipitate after sonication, incubation and ammonium sulphate salting-out at 0-10% saturation; 10 - dialysis I supernatant; 11 - dialysis I sediment, and in chapter B, the numbers mean: 1 - LMW protein mass standard; 2 - input for the Macro-Prep High Q Media bed; 3 - fractions not bound to the Macro-Prep High Q Media bed; 4 - fractions bound to the Macro-Prep High Q Media bed; 5 - fractions entering the Pierce High- Capacity Endotoxin Removal Resin deposit; 6 - fractions eluted from High-Capacity Endotoxin Removal Resin; 7,8,9,10 - final protein solution for lyophilization.

Fig. 14. Shows a graph of L929 cell proliferation, depending on the type of culture vessel coating. Amount of fluorescence in alamarBlue staining of L929 cells on plates coated with RE15mR protein, compared to fibronectin.

Fig. 15. Shows a graph of L929 cell proliferation, depending on the type of culture vessel coating. Amount of fluorescence in alamarBlue staining of L929 cells on plates coated with EJ17zipR protein, compared to fibronectin.

Fig. 16. Shows a graph of L929 cell adhesion depending on the protein used for coating and its concentration. Amount of fluorescence in L929 cell culture on plates coated with RE15mR protein, compared to fibronectin. Fig. 17. Shows a graph of L929 cell adhesion depending on the protein used for coating and its concentration. Amount of fluorescence in culture of L929 cells on plates coated with EJ17zipR protein, compared to fibronectin.

Fig. 18. Shows a graph of RE15mR protein cytotoxicity against L929 cells - measurement of absorbance in the MTT assay.

Fig. 19. Shows the cytotoxicity of theEJ17zipR protein against L929 cells - measurement of absorbance in the MTT assay.

The invention is related to recombinant structural proteins obtained by genetic engineering using an eukaryotic or prokaryotic expression system, preferably the epithelium of the Chinese hamster ovary CHO or Escherichia coli. The recombinant structural proteins described in the invention are composed of domains of the structural proteins resilin, elastin and silk fibroin. Preferably, the resilin domains in the invention are represented by the sequences: SDTYGAPGGGNGGRP, GGRPSDSYGAPGGGN, GGRPSDSF(or M)GAPGGGN, PGGGNGGRPSDTYGA, GGRPSSSYGAPGQGN, GGRPSDSFGAPGGGN, GAPAQTPSSQY, AQTPSSQYGAP; preferably GGRPSDSYGAPGGGN. Elastin domains are preferably represented by the sequences: VPGXG (wherein X = V, I, A) or JGGZG (wherein J, Z = V, L, A); most preferably VPGIG and VPGAG. Silk fibroin domains are preferably represented by the sequences: GAGAGS. The recombinant hybrid proteins of the invention are enriched with functional domains, preferably:

- the RGD motif of the fibronectin cell adhesion sequence; most preferably AVTGRGDSPASS, optionally a shortened GRGDSP or an extended TVYAVTGRGDSPASS;

- an MMP motif recognized by matrix metalloproteinases, most preferably GPQGIWGQ;

- lysine-rich K+ cross-linking domains enabling chemical modifications of recombinant structural proteins; most preferably GGKGGKGGKGG;

- a ZIP domain encoding the stabilizing supramolecular structure of the leucine zipper; most preferably

VGGGGGKENQIAIRASFLEKENSALRQEVADLRKELGKCKNILAKYEAGGGGG. Without departing from the scope of the present invention, one skilled in the art can make quantitative or qualitative changes to the functional domains to adjust the properties of the recombinant hybrid proteins of the invention.

Enriching the sequence of recombinant hybrid proteins with RGD domains binding integrins, derived from fibronectin, promotes the adhesion of many types of endothelial cells, smooth muscle cells and fibroblasts, thanks to which biomaterials enriched with recombinant hybrid proteins have a positive effect on the growth of cells having physical contact with them. Metalloproteinase-sensitive MMP sequences, derived from the human alpha(I) collagen chain, were added to recombinant structural proteins to promote proteolytic degradation associated with the possibility of extracellular matrix rearrangement by proliferating cells. The presence of the K+ motif is intended to functionalize the peptides with selected chemical groups to facilitate the controlled crosslinking of biomaterials enriched with recombinant hybrid proteins, desired in bioprinting. Enrichment of recombinant structural proteins with a ZIP sequence from the hepatic leukemia factor (HLF) dimerization domain is expected to provide structural stability during printing, due to the ability to form amphiphilic alpha helical structures based on hydrophobic interactions.

According to the invention, the use of the described domain combinations allows to use the developed protein in the polymeric biomaterial remodelling by cells, but also due to its general importance as a collagenase substrate: to mediate cell invasion, migration, proliferation, and growth of new tissues. The obtained recombinant hybrid proteins will enable modulating cell growth by fine-tuning the RGD density and introducing MMP- sensitive domains in the created hydrogels, which are to simulate the extracellular matrix (ECM).

Example 1

According to the invention, the recombinant RE15mR hybrid protein is composed sequentially of 3 resilin domains, 1 K+ domain, 1 MMP domain, 3 elastin domains, 1 K+ domain, 4 elastin domains, 1 K+ domain, 4 elastin domains, 1 K+ domain, 3 elastin domains , 1 MMP domain, 7 RGD domains. In a preferred embodiment, the protein in the amino acid sequence comprises about 12-15% proline and about 32-38% glycine. In another preferred embodiment, the resulting full-length protein should have a molecular weight of about 26 kDa. Figure 1 shows a diagram of the RE15mR recombinant hybrid protein which is another preferred embodiment. Figure 4 shows a preferred embodiment of the DNA sequence encoding the RE15mR recombinant hybrid protein. Figure 6 shows the most preferred embodiment of the amino acid sequence of the RE15mR recombinant hybrid protein of the invention.

Example 2

According to the invention, the recombinant hybrid protein EJ17zipR is composed sequentially of 1 ZIP domain, 56 elastin domains, 9 silk fibroin domains, 7 RGD domains, 56 elastin domains, 10 silk fibroin domains, 7 RGD domains. In a preferred embodiment, the protein in the amino acid sequence comprises about 12-17% proline and 20-30% glycine. In another preferred embodiment, the resulting full-length protein should have a molecular weight of about 86 kDa. The protein should oligomerize into molecules above 1 mDa (verification method - polyacrylamide gel electrophoresis under native conditions). Figure 2 shows a diagram of the recombinant hybrid protein EJ17zipR, constituting another preferred embodiment of the invention. Figure 5 shows a preferred embodiment of the DNA sequence encoding the recombinant EJ17zipR hybrid protein of the invention. Figure 7 shows the most preferred embodiment of the amino acid sequence of the recombinant EJ17zipR hybrid protein of the invention.

Example 3

In a preferred embodiment, plasmid vectors containing a promoter derived from the T7 phage, preferably pETlla-d, pET15b, pET19b, pET28a-c(+), pET21a-d(+), pET22b(+), were used to obtain the recombinant hybrid proteins of the invention. pET23a-d(+), pEt25b(+), pET44a-c(+), pET46Ek/LIC, most preferably pETl la. Sequences encoding the recombinant hybrid proteins of the invention, such as those shown in Figure 4 or 5, were incorporated into the vector using molecular cloning methods known in the art using selected restriction sites. In a preferred embodiment, the restriction sites were Ndel and BamHI. The cloned sequences were amplified using the engineered primers listed in Figure 3. An expression vector enabling efficient and stable expression of recombinant proteins in E. coli cells was obtained. In a preferred embodiment, the E. coli strain was BLR(DE3). The preferred embodiment of an expression vector encoding the RE15mR recombinant hybrid protein is shown in Figure 8. The preferred embodiment of the expression vector encoding the EJ17zipR recombinant hybrid protein is shown in Figure Example 4

The invention relates to a method for producing recombinant hybrid proteins RE15mR and EJ17zipR. Recombinant DNA sequences encoding recombinant hybrid proteins of the invention, vectors comprising recombinant DNA sequences of the invention, can be used in the eukaryotic or prokaryotic expression systems of the invention to produce recombinant RE15mR and EJ17zipR hybrid proteins. In a preferred embodiment, the eukaryotic or prokaryotic expression systems were CHO Chinese hamster ovary epithelial cells or Escherichia coli cells. A person skilled in the art will appreciate the wide selection of expression systems. Eukaryotic or prokaryotic expression systems mean the so-called protoplasts derived from the foregoing expression systems.

In a preferred embodiment, the method comprises the following steps:

1. Culture of E. coli bacterial cells of the BLR(DE3) strain, optionally BL21(DE3), transformed with a plasmid encoding recombinant hybrid proteins, with the addition of an appropriate antibiotic (preferably ampicillin at 50-200 pg/mL). The method according to the invention uses conventional culture media, selected according to the host strain used. For the BL21(DE3) and BLR(DE3) strains used, it can be a standard, rich LB medium, additionally supplemented with proline and glycine. There is a mineral medium used in bioreactor cultures, and the composition of said medium was originally developed during the experiments leading to the development of the invention. This is the medium the composition of which is shown in Figure 10. Growing cultures using this medium, especially large-scale cultures in a bioreactor, presents the advantage of relatively low cost, while ensuring a satisfactory growth level (comparable to the level of growth in media used in laboratory-scale cultures). Bacterial biomass is produced In the first phase of culture, and culture parameters for this phase are: temperature: 30 °C, agitation: 150-700 rpm, aeration: 5-10 LPM, DO: >20%, pH: 7.1 +/-1. Culture is carried out until the optical density OD600 of 0.7-0.9 is reached. Next, the induction of expression of recombinant hybrid proteins is carried out by adding IPTG isopropyl-P-D-1 -thiogalactopyranoside to a concentration of 0.4-1.0 mM, or lactose to a concentration of 5-20 mM, and the culture is carried out under the following conditions: temperature: 37 °C , agitation: 500-700 rpm, aeration: 7-10 LPM, DO: >20%, pH: 7.1 +/-1 for 5-7 hours until an optical density of OD600 3.0 - 9.0 is reached. 2. At this stage, the bacterial biomass is separated from the culture medium by centrifugation. The bacterial cells are then suspended in a lysis buffer formulated for this purpose, shown in Figure 11. From the invention perspective, it is preferable to have bacterial biomass disintegration carried out using a high-pressure flow disintegrator, with a set pressure of 800-900 bar. Over the course of work, it is preferable to perform 3 to 4 disintegration rounds and add 0.4% (w/v) polyethyleneimine (PEI) to the suspension to precipitate the host DNA.

3. Separation of insoluble protein fractions, non-disintegrated bacterial cells and precipitated DNA from the supernatant containing recombinant hybrid proteins by centrifugation.

4. Incubation of the supernatant containing the recombinant hybrid proteins at 90°C for 15-30 min, and centrifugation of the denatured proteins. During the conducted experiments, it was also shown that it is advantageous to add a protease inhibitor cocktail to the recombinant protein solution at this stage (cOmplete, EDTA-free, Roche, Cat. 05056489001) in the amount of 1 tablet per 50 mL of solution.

5. Precipitation of recombinant protein from solution by adding ammonium sulfate to 10% saturation (for EJ17zipR protein) or 20-40% saturation (for RE15mR protein) at room temperature, followed by centrifugation of precipitated proteins and dissolution in 20-40 mM TRIS buffer with 10 mM EDTA pH 8.0.

6. Dialysis of the recombinant protein suspension into 20-40 mM TRIS buffer with 10 mM EDTA pH 8.0 for 24-48 hours at 4 °C.

7. Protein purification on Macro-Prep High Q Media (Bio-Rad). The bed-filled column was equilibrated with a calibration buffer with the following composition: 20-50 mM TRIS buffer pH 8.0. The protein solution obtained as a result of salted protein dissolution was applied to the column equilibrated as such. The separation was carried out in the FPLC system. Proteins not bound to the matrix were washed away with the calibration buffer. The proteins bound to the bed were eluted with an elution buffer composed of 20-50 mM TRIS buffer pH 8.0 + 1 M NaCl. A flow of 1-2 mL/min was used during the separation, and fractions above the absorbance of 0.05 AU were collected. The concentration of eluted protein was determined by the Bradford and BCA method (Pierce BCA protein Assay Kit). According to the invention, the recombinant hybrid proteins RE15mR and EJ17zipR did not bind to the matrix and were eluted from the column with the calibration buffer.

8. Endotoxin removal using Pierce High-Capacity Endotoxin Removal Resin columns according to the manufacturer's instructions.

9. Dialysis of the recombinant protein suspension to ddH2O for 24h-48h at 4 °C.

10. Lyophilization of recombinant hybrid protein.

The method is characterized in that the obtained recombinant RE15mR protein is able to gel at a concentration above 200 mg/mL at 4 °C, with the addition of 0.5M NaCl. The recombinant EJ17zipR protein does not form hydrogels. The yield of the method described in the invention is 20-70 mg of recombinant RE15mR and EJ17zipR protein from 1 litre of culture. The course of the purification process for the recombinant RE15mR structural protein in terms of the product profile, compared to impurities in SDS-PAGE electrophoresis under denaturing conditions, is shown in Figure 12. The course of the purification process for the recombinant structural protein EJ17zipR in terms of the product profile, compared to impurities in SDS-PAGE electrophoresis under denaturing conditions is shown in Figure 13.

Example 5

In another preferred embodiment, the obtained recombinant hybrid proteins according to the invention were subjected to a methacrylation process. In a preferred embodiment, the method comprises the following steps:

1. Recombinant hybrid protein according to the invention from the batch to be subj ected to the methacrylation process was weighed into the reaction vessel with a capacity of X mL on an analytical balance.

2. The reaction vessel was equipped with a stir bar and placed over a magnetic stirrer. Using an automatic pipette, X mL of PBSxl buffer was metered into it to obtain a 2% (w/v) solution. The rotation of the stirrer was set in the range of 200 to 1500 rpm, with about 1000 rpm in the preferred embodiment. The mixture was left under constant stirring at room temperature until the starting material was completely dissolved.

3. After complete dissolution of the substrate, the reaction vessel was placed in a water- ice bath (a crystallizer filled with water and ice to half its volume so as to maintain the lability of the liquid phase) and protected from light. Expected bath temperature < 4 °C.

4. Using an automatic pipette, X ml of methacrylic anhydride (MMA) or other electrophilic reagent was measured and added to the reaction.

In the case of the protein methacrylation reaction, it is necessary to select the appropriate amount of methacrylic anhydride each time, taking the series of a given protein into account. In order to do this, read the amino acid sequence of the protein and read how many lysine residues are in the protein chain. Next, taking into account the total mass of 1 mole of protein and the mass weighed for the reaction, calculate how much methacrylic anhydride should be used with the ratio of free amino groups of lysine to anhydride equal ton _NH2 , : n _MMA = 1: 1 . The following formula should be used on the calculations: mprotein . _M

¹ M ‘‘lysine ¹ ‘MM A ‘protein

^V MMA ■ 1000 d-MMA wherein: v _MMj4 — methacyrlic anhydride volume [pl]

The amount of methacrylic anhydride used is a parameter that controls the degree of substitution. Depending on the expected degree of methacrylation, it is possible to use a 1- to 10-fold excess of the anhydride relative to the free amino groups of the lysine. For this purpose, the anhydride volume calculated from the foregoing formula should be multiplied by a number from the range of 1-10.

In addition to lysine amino groups, other amino acid units, such as tryptophan, tyrosine, lysine, threonine, serine or arginine, may also undergo methacrylation, as long as they are present in the amino acid sequence. When calculating the degree of protein substitution, remember to take them into account. 5. The reaction was carried out under given conditions (T < 4°C, stirring in the range from 200 to 1500 rpm, at about 1000 rpm in the preferred embodiment) to react, preferably for 24 hrs.

6. After the time required for the reaction had elapsed, the PBSxl solution was added in portions to the mixture, until a 5-fold dilution of the post-reaction mixture was obtained (1 :4 mixture :PBSxl).

7. The obtained solution was poured into dialysis tubes, and the dialysis process was carried out at a temperature of preferably 3-8°C, at 4°C in the preferred embodiment, to complete reaction, preferably for 2 days, changing the water, preferably once a day.

8. After the dialysis process was completed, the solution from the dialysis tubes was quantitatively transferred to a large beaker, and then approx. 5 mL each (using an automatic pipette) to glass vials.

Note: Each empty glass vial should be weighed and its mass reported as mi. Then, transfer the solution to it, weigh it again, and describe the mass as m2.

9. The solution vials were placed in a -80°C freezer and frozen, preferably for a minimum of 3 hours. Next, it was lyophilized according to the following parameters: a) shelf temperature: 0°C b) pressure: 0.100 mbar c) duration: 48 hrs

10. The vials with the obtained lyophilisates were weighed and the mass was described as m3. Then, Am=m3-mi was calculated to determine the weight of the lyophilisate in each vial.

11. The vials with the material were stored at -20°C.

Example 5

The biological activity of the recombinant hybrid proteins of the invention was tested, including their effect on cell viability, proliferation, adhesion and cytotoxicity. Protocols, reagents and equipment routinely used in this type of research were used. The control in

SUBSTITUTE SHEET (RULE 26) the study was provided by fibronectin applied as coating in the amount of 1 pg/cm ² . The negative control was provided by the absence of any protein coating in the well.

Cell proliferation and adhesion

The prepared plates coated with the recombinant hybrid proteins of the invention were plated with L929 cells at 5xlO ³ /well. Cells were incubated in dedicated culture medium for 2, 24 and 48 hours. After this time, the culture wells were rinsed with sterile phosphate-buffered saline (PBS buffer) to remove dead and non-adherent cells, cells were stained with alamarBlue for three hours, and fluorescence was measured at 530 nm (excitation) and 590 nm (emission) wavelengths.

Increase in fluorescence after 48 hours vs. 2 and 24 hours, taking into account the division time for the L929 cell line (Fig. 14 for RE15mR and Fig. 15 for EJ17zipR) indicates active cell proliferation in both the positive control, and wells coated with the test protein. It can therefore be concluded that the RE15mR protein does not interfere with the cell proliferation rate used in the cell line study.

Analogously to the foregoing conclusions, the increase in fluorescence during incubation (Fig. 16 for RE15mR and Fig. 17 forEJ17zipR) indicates cell adhesion to the plastic used in both the positive control and wells coated with the test protein. It was observed that the RE15mR protein promotes the adhesion of cells used in the study of the cell line to an extent comparable to fibronectin - a commercially available protein used to coat surfaces intended for eukaryotic cell culture. It was observed that the EJ17zipR protein promotes the adhesion of cells used in the cell line study, although the number of adhering cells is lower compared to fibronectin. The effect of the concentration of the protein used on the adhesion of the cells used is clearly visible - the number of adhering cells increases with the increase in the protein concentration after 24 hours of culture.

Cytotoxicity

Cytotoxicity of RE15mR and EJ17zipR proteins against L-929 cells (measurement of absorbance in the MTT assay) was tested according to ISO 10993-5:2009(E): Biological Evaluation of Medical Devices. Part 5: Tests for in vitro cytotoxicity. Depending on the planned exposure time, cells were plated in 96-well plates at densities of lxl0 ⁵ /mL, 5xlO ⁴ /mL and 2.5xlO ⁴ /mL, respectively, 100 pL per well. Culture was carried out overnight under standard conditions (5% CO2 and 37 °C) in supplemented DMEM medium, so that the fibroblasts had a chance to adhere to the bottom of the culture vessel. The study was performed with the direct method by adding a solution of purified protein reconstituted in the medium to the culture. After checking the confluence and population status, the protein solution in the medium was applied to the plates in the amount of 100 pL, at the concentration of 1, 0.5 and 0.1 mg/mL. The plate with cells at a density of lxlO ⁵ /mL was incubated for 24 hours, while the culture at a starting density of 5xlO ⁴ /mL was exposed to RE15mR protein for 48 hours. In contrast, the plate with cells at a density of lxlO ⁵ /mL was incubated for 24 hours, the culture at a starting density of 5xlO ⁴ /mL was exposed to Ej 17zipR protein in the medium for 48 hours, and cells with a starting density of 2.5xlO ⁴ /mL were exposed to EJ17zipR solutions for 72 hours. After this time, the cells were incubated for 2 hours with the MTT reagent solution, then all the cell supernatant was removed and the formed formazan salt crystals were dissolved in DMSO. The amount of coloured product formed, proportional to the number of viable cells, was determined by measuring the absorbance at 570 and 650 nm wavelengths. According to the standard, the expected result is the viability of cells exposed to the cytotoxic agent not less than 70% of the viability of untreated cells in the negative control.

Based on the obtained results, it was found that RE15mR and EJ17zipR proteins in the tested concentration range (0.1 - 1 mg/mL) with the foregoing exposure are not cytotoxic to the L-929 fibroblast line (Fig. 18 for RE15mR protein Fig. 19 for the EJ17zipR protein).