Recombinant protein, recombinant DNA sequence, vector, eukaryotic and prokaryotic expression system and application thereof

Abstract

The invention relates to a recombinant RE15mR and EJ17zipR hybrid protein with a structure as described in the specification, a recombinant DNA sequence for coding the recombinant hybrid protein, a vector containing the recombinant DNA sequence, and a eukaryotic or prokaryotic expression system transformed by using the recombinant DNA sequence. The invention also relates to the use of the recombinant hybrid protein as a component of bio-inks intended for bio-printing and as a component of biological materials. In addition, the invention also relates to the use of the recombinant DNA sequence, the vector and the eukaryotic or prokaryotic expression system for preparing the recombinant hybrid protein.

Description

Technical Field

[0001] This invention relates to a recombinant RE15mR and EJ17zipR hybrid protein having the structure described in the specification, a recombinant DNA sequence encoding the recombinant hybrid protein, a vector containing the recombinant DNA sequence, and eukaryotic and prokaryotic expression systems transformed with the recombinant DNA sequence. The invention also relates to the use of the recombinant hybrid protein as a component of bioinks intended for use in bioprinting and as a component of biomaterials. Furthermore, the invention relates to the use of the recombinant DNA sequence, vector, and eukaryotic or prokaryotic expression system for the preparation of the recombinant hybrid protein. This invention can be used in bioprinting, medicine, and cell and tissue engineering. Background Technology

[0002] Bioprinting and biomaterials are currently under extensive research and development. Technological solutions in these fields enable the 3D printing of tissue structures and their eventual application in regenerative medicine. Therefore, examples of natural and synthetic bioinks and biomaterials already exist in this field, such as the biopolymers disclosed in US Patent 2022 / 0047706. However, the materials used in bioinks and biomaterials still require improvement in their effects on cell viability, proliferation, adhesion, or cytotoxicity (which may also be incorporated into blends).

[0003] Purpose of the invention

[0004] The object of this invention is to provide a novel, alternative recombinant hybrid protein, particularly suitable as a component of bioinks and biomaterials. This protein should be versatile, safe, and effective for both laboratory settings and ultimately for patient use. Furthermore, the invention aims to overcome the technical problems found in the prior art, particularly by providing a protein that is equivalent to or superior to proteins currently used in the prior art in at least one parameter, such as its effects on cell viability, proliferation, adhesion, or cytotoxicity (which may also be incorporated into blends), rheological parameters (composite modulus, dynamic viscosity), printability (fiber printability, resolution, continuity, and stability), and mechanical properties (compression strength, Young's modulus). Summary of the Invention

[0005] The first aspect of the present invention relates to a recombinant RE15mR hybrid protein, which comprises a sequence of three arthropod elastin domains, one K+ domain, three arthropod elastin domains, one MMP domain, three elastin domains, one K+ domain, four elastin domains, one K+ domain, three elastin domains, one K+ domain, four elastin domains, one K+ domain, three elastin domains, one MMP domain, and seven RGD domains.

[0006] Preferably, the amino acid sequence of the recombinant RE15mR hybrid protein according to the present invention contains 12-15% proline and / or 32-38% glycine.

[0007] Preferably, the molecular weight of the recombinant RE15mR hybrid protein of the present invention is 26 kDa.

[0008] Preferably, the recombinant RE15mR hybrid protein of the present invention has the amino acid sequence of SEQ #3.

[0009] Preferably, the recombinant RE15mR hybrid protein of the present invention is a fragment of a recombinant protein.

[0010] Preferably, the recombinant RE15mR hybrid protein of the present invention is at least partially methacrylamide.

[0011] In a second aspect, the present invention provides a recombinant DNA sequence selected from:

[0012] The recombinant DNA sequence encodes the recombinant RE15mR heterozygous protein as defined above.

[0013] The recombinant DNA sequence contains a region encoding a DNA sequence encoding the recombinant RE15mR heterozygous protein as defined above, and

[0014] The recombinant DNA sequence hybridizes with the DNA sequence encoding the recombinant heterozygous protein RE15mR as defined above.

[0015] Preferably, the recombinant DNA sequence of the second aspect of the present invention has a sequence that is at least 80% identical to, more preferably at least 90% identical to, and most preferably homologous to the recombinant DNA sequence of SEQ #1.

[0016] In a third aspect, the present invention provides a vector comprising a recombinant DNA sequence comprising a region of a recombinant DNA sequence according to a second aspect of the present invention, and / or a recombinant sequence comprising a portion of a region of a recombinant DNA sequence according to a second aspect of the present invention. Preferably, the vector according to the present invention is a plasmid containing a promoter derived from bacteriophage T7, preferably selected from pET11a-d, pET15b, pET19b, pET28a-c(+), pET21a-d(+), pET22b(+), pET23a-d(+), pET25b(+), pET44a-c(+), pET46Ek / LIC, most preferably pET11a.

[0017] In a fourth aspect, the present invention provides a eukaryotic or prokaryotic expression system transformed with a recombinant DNA sequence and / or a recombinant sequence comprising a region containing a portion of a recombinant DNA sequence comprising a portion of a region containing a recombinant DNA sequence comprising a portion of ... recombinant DNA sequence comprising a portion of a recombinant DNA sequence comprising a recombinant DNA sequence comprising a portion of a recombinant DNA sequence comprising a recombinant DNA sequence comprising a recombinant DNA sequence comprising a recombinant DNA sequence comprising a recombinant DNA sequence comprising a recombinant DNA sequence comprising a recombinant DNA sequence comprising a recombinant DNA sequence comprising a recombinant DNA sequence comprising a recombinant DNA sequence comprising a recombinant DNA sequence comprising a recombinant DNA sequence comprising a recombinant DNA sequence comprising a recombinant DNA sequence comprising a recombinant DNA sequence comprising a recombinant DNA sequence comprising a recombinant DNA sequence comprising a recombinant DNA sequence comprising a recombinant DNA sequence comprising a recombinant DNA sequence comprising a recombinant DNA sequence comprising a recom

[0018] In the context of this invention, the term "eukaryotic or prokaryotic expression system" also includes so-called protoplasts derived from such expression system.

[0019] In a fifth aspect, the subject of the present invention is a recombinant hybrid protein EJ17zipR, which consists of one ZIP domain, 56 elastin domains, 10 silk fibroin domains, and 7 RGD domains.

[0020] Preferably, the amino acid sequence of the recombinant EJ17zipR hybrid protein of the present invention contains 12-17% proline and / or 20-30% glycine.

[0021] Preferably, the recombinant hybrid protein EJ17zipR according to the present invention has a molecular weight of 86 kDa.

[0022] Preferably, the recombinant EJ17zipR hybrid protein of the present invention has the amino acid sequence of SEQ #4.

[0023] Preferably, the recombinant EJ17zipR hybrid protein of the present invention is a recombinant protein fragment.

[0024] Preferably, the recombinant EJ17zipR heterozygous protein of the present invention is at least partially methacrylamide.

[0025] In a sixth aspect, the present invention provides a recombinant DNA sequence selected from:

[0026] The recombinant DNA sequence encodes the recombinant heterozygous protein EJ17zipR as defined above.

[0027] The recombinant DNA sequence contains a region encoding a DNA sequence that encodes the recombinant heterozygous protein EJ17zipR as defined above, and

[0028] The recombinant DNA sequence hybridizes with the DNA sequence encoding the recombinant heterozygous protein EJ17zipR as defined above.

[0029] Preferably, the recombinant DNA sequence of the sixth aspect of the present invention has a sequence that is at least 80% identical, more preferably at least 90% identical, and most preferably homologous to the recombinant DNA sequence of SEQ #2.

[0030] In a seventh aspect, the present invention provides a vector comprising a recombinant DNA sequence and / or a recombinant sequence, said recombinant DNA sequence containing a region comprising the recombinant DNA sequence according to a sixth aspect of the present invention, said recombinant sequence containing a portion of the region comprising the recombinant DNA sequence according to a sixth aspect of the present invention. Preferably, the vector according to the present invention is a plasmid containing a promoter derived from bacteriophage T7, preferably selected from pET11a-d, pET15b, pET19b, pET28a-c(+), pET21a-d(+), pET22b(+), pET23a-d(+), pEt25b(+), pET44a-c(+), pET46Ek / LIC, most preferably pET11a.

[0031] In an eighth aspect, the present invention provides a eukaryotic or prokaryotic expression system transformed with a recombinant DNA sequence and / or a recombinant sequence comprising a region including the recombinant DNA sequence according to a sixth aspect of the present invention, wherein the recombinant sequence comprises a portion of the region including the recombinant DNA sequence according to a sixth aspect of the present invention. Preferably, the expression system is selected from Chinese hamster ovarian epithelial (CHO) cells and Escherichia coli cells.

[0032] In a ninth aspect, the present invention provides the use of a recombinant hybrid protein selected from the RE15mR protein according to the first aspect of the invention and the EJ17zipR protein according to the fifth aspect of the invention as a component of a bioprinting ink.

[0033] In a tenth aspect, the present invention provides the use of a recombinant hybrid protein selected from the RE15mR protein according to the first aspect of the invention and the EJ17zipR protein according to the fifth aspect of the invention as a component of a biomaterial.

[0034] In an eleventh aspect, the present invention provides the use of a recombinant DNA sequence according to the second aspect of the present invention, or a vector according to the third aspect of the present invention, or a eukaryotic or prokaryotic expression system according to the fourth aspect of the present invention, for preparing a recombinant RE15mR hybrid protein according to the first aspect of the present invention.

[0035] In a twelfth aspect, the present invention provides the use of a recombinant DNA sequence according to the sixth aspect of the present invention, or a vector according to the seventh aspect of the present invention, or a eukaryotic or prokaryotic expression system according to the eighth aspect of the present invention, for preparing the recombinant hybrid protein EJ17zipR according to the fifth aspect of the present invention.

[0036] The two proteins according to the present invention, namely recombinant hybrid proteins RE15mR and EJ17zipR, are both chimeric proteins containing sequences derived from elastin, i.e., replicating the native elastin pentamer. Therefore, both proteins are classified as elastin-like proteins (ELPs).

[0037] Invention Effects

[0038] This invention relates to bioprinting processes. The prepared proteins can be additives to natural bioinks (e.g., based on decellularized extracellular matrix (dECM); mixtures of single proteins / natural compounds) and synthetic bioinks, serving as additives to improve cell survival, proliferation, and adhesion. Due to the independent structural domains in the proteins designed according to this invention, they can also be used as basic components of bioinks (not just as additives as described above).

[0039] The proteins according to the present invention, due to their structure (mainly collagen and elastin domains), can be used in a broad sense of regenerative medicine, a field that also uses other technologies (other than 3D bioprinting) to prepare biomaterials.

[0040] In addition to being used in the preparation of bio-inks and biomaterials for biomimetic organs and tissue systems, the proteins according to the present invention can also be used in regenerative medicine, for example as medical dressings or components thereof.

[0041] The protein according to the invention will be used to manufacture biomaterials in both methacrylated and non-methacrylated forms, which will undoubtedly expand the possibilities of its use in the final product.

[0042] The proteins according to the present invention have many benefits, the most important of which are, for example, improving the stiffness, degradation rate, elasticity or flexibility of printed products by improving cell adhesion and growth, and improving the physicochemical and biological properties of bioprinted 3D tissue models, biomimetic organs and biomaterials for scientific research and regenerative medicine. Attached Figure Description

[0043] The subject matter of the invention in the embodiments is illustrated in the accompanying drawings in a manner that does not limit the scope of the invention, wherein:

[0044] Figure 1 A schematic diagram of the RE15mR recombinant hybrid protein is shown;

[0045] Figure 2 A schematic diagram of the EJ17zipR recombinant hybrid protein is shown;

[0046] Figure 3 A primer sequence listing for the gene encoding the recombinant heterozygous protein is shown;

[0047] Figure 4The DNA sequence encoding the RE15mR recombinant heterozygous protein is shown in SEQ#1;

[0048] Figure 5 The DNA sequence SEQ#2 encoding the EJ17zipR recombinant heterozygous protein is shown.

[0049] Figure 6 The amino acid sequence of the RE15mR recombinant hybrid protein is shown;

[0050] Figure 7 The amino acid sequence of the EJ17zipR recombinant hybrid protein is shown;

[0051] Figure 8 A map of the expression vector carrying the cloned gene encoding the RE15mR heterozygous protein is shown;

[0052] Figure 9 A map of the expression vector carrying the cloned gene encoding the EJ17zipR heterozygous protein is shown;

[0053] Figure 10 shows SDS-PAGE separation images of samples collected in subsequent stages from which the recombinant heterozygous protein RE15mR was obtained;

[0054] Figure 11 shows SDS-PAGE separation images of samples collected in subsequent stages from the acquisition of the recombinant heterozygous protein EJ17zipR;

[0055] Figure 12A The RE15mR protein before methacrylation was shown. 1 H NMR spectrum;

[0056] Figure 12B The RE15mR protein after methacrylation was shown. 1 H NMR spectrum;

[0057] Figure 12C The 1H NMR spectrum of the methacrylated RE15mR protein (from batch 15_E_003) is shown.

[0058] Figure 12D The EJ17mR protein before methacrylation was shown. 1 H NMR spectrum;

[0059] Figure 12E The methacrylated EJ17mR protein was shown. 1 H NMR spectrum;

[0060] Figure 13AThe fluorescence level of AlamarBlue staining of L929 cells on RE15mR protein-coated plates versus fibronectin-coated plates is shown in the L929 cell proliferation assay.

[0061] Figure 13B The fluorescence level of AlamarBlue staining of HUVEC cells on RE15mR protein-coated plates versus fibronectin-coated plates is shown in the HUVEC cell proliferation assay.

[0062] Figure 14A The fluorescence levels of L929 cell cultures on RE15mR protein-coated plates compared to fibronectin-coated plates were shown in the L929 cell adhesion assay.

[0063] Figure 14B The fluorescence levels of HUVEC cultures on RE15mR protein-coated plates compared to fibronectin-coated plates were shown in the HUVEC cell adhesion assay.

[0064] Figure 15 The fluorescence level of L929 cell cultures on RE15mR protein-coated plates compared to fibronectin-coated plates was shown in the RE15mR protein cytotoxicity assay for L929 cells.

[0065] Figure 16 Photographs of samples from HUVEC and HDFα cells stained with calcein AM and ethidium homodimer at 24, 48 and 96 hours after exposure to RE15mR protein contaminants are shown.

[0066] Figure 17 The absorbance measurements in the MTT assay are shown in the study of RE15mR protein cytotoxicity against L929 reference cells.

[0067] Figure 18A The fluorescence level of AlamarBlue staining of L929 cells on EJ17zipR protein-coated plates versus fibronectin-coated plates is shown in the L929 cell proliferation assay.

[0068] Figure 18B The fluorescence level of AlamarBlue staining in HUVEC cells on EJ17zipR protein-coated plates versus fibronectin-coated plates is shown in the HUVEC cell proliferation assay.

[0069] Figure 19A The fluorescence levels of L929 cell cultures on EJ17zipR protein-coated plates compared to fibronectin-coated plates were shown in the L929 cell adhesion assay.

[0070] Figure 19B The fluorescence levels of HUVEC cultures on EJ17zipR protein-coated plates versus fibronectin-coated plates were shown in the HUVEC cell adhesion assay.

[0071] Figure 20 The fluorescence levels of L929 cell cultures on EJ17zipR protein-coated plates compared to fibronectin-coated plates were shown in the cytotoxicity assay of EJ17zipR protein against L929 cells.

[0072] Figure 21 Photographs of samples from HUVEC and HDFα cells stained with calcein AM and ethidium homodimer at 24, 48, and 96 hours after exposure to contaminants containing EJ17zipR protein are shown.

[0073] Figure 22 The absorbance measurements from the MTT assay are shown in the study of the cytotoxicity of EJ17zipR protein against L929 reference cells.

[0074] Figure 23 Rheological measurements of GelMa-based test materials containing RE15mR recombinant protein are shown, where A-gel point, B-composite modulus, and C-dynamic viscosity.

[0075] Figure 24 The results of evaluating the printability of GelMa-based biomaterials containing the RE15mR recombinant protein are shown, including the results of the A-fiber fusion test and the results of the B-fiber collapse test.

[0076] Figure 25 The mechanical parameters of a 10% GelMa-based bioink containing RE15mR protein are shown, where A - mechanical strength, B - Young's modulus, and C - conventional yield strength.

[0077] Figure 26 The results of water absorption determination for GelMa-based materials containing RE15mR recombinant protein are shown.

[0078] Figure 27 The rheological measurements of the test material containing dECM and RE15mR proteins are shown, where A - gel point, B - composite modulus, and C - dynamic viscosity.

[0079] Figure 28 The printability assessment results of biomaterials containing dECM and RE15mR recombinant proteins are shown, including A-fiber fusion test results and B-fiber collapse test results;

[0080] Figure 29The mechanical parameters of the bio-ink containing dECM and recombinant RE15mR protein are shown, where A - mechanical strength, B - Young's modulus, and C - conventional yield strength.

[0081] Figure 30 The results of water absorption determination for test materials containing dECM and RE15mR proteins are shown.

[0082] Figure 31 Rheological measurements of GelMa-based test materials containing recombinant EJ17zipR protein are shown, where A-gel point, B-composite modulus, and C-dynamic viscosity.

[0083] Figure 32 The printability assessment results of GelMa-based biomaterials containing the EJ17zipR recombinant protein are shown, including A-fiber fusion test results and B-fiber collapse test results;

[0084] Figure 33 The mechanical parameters of a 10% GelMa-based bioink containing EJ17zipR protein are shown, where A - mechanical strength, B - Young's modulus, and C - conventional yield strength.

[0085] Figure 34 The water absorption of GelMa-based materials containing recombinant EJ17zipR protein is shown in the figure.

[0086] Figure 35 The rheological measurements of the test material containing dECM and EJ17zipR proteins are shown, where A - gel point, B - composite modulus, and C - dynamic viscosity.

[0087] Figure 36 The printability assessment results of biomaterials containing dECM and EJ17zipR proteins are shown, including A-fiber fusion test results and B-fiber collapse test results;

[0088] Figure 37 The mechanical parameters of the bio-ink containing dECM and EJ17zipR proteins are shown, where A - mechanical strength, B - Young's modulus, and C - conventional yield strength.

[0089] Figure 38 The results of water absorption determination of the test material containing dECM and EJ17zipR proteins are shown. Detailed Implementation

[0090] This invention relates to recombinant structural proteins obtained through genetic engineering using eukaryotic or prokaryotic expression systems (preferably Chinese hamster ovarian CHO epithelium or *Escherichia coli*). The recombinant structural proteins according to the invention consist of domains of the structural proteins arthropod elastin, elastin, and silk fibroin. Preferably, the arthropod elastin domain of the invention is represented by the following sequences: SDTYGAPGGGNGGRP, GGRPSDSYGAPGGGN, GGRPSDSF (or M)GAPGGGN, PGGGNGGRPSDTYGA, GGRPSSSYGAPGQGN, GGRPSDSFGAPGGGN, GAPAQTPSSQY, AQTPSSQYGAP, and most preferably GGRPSDSYGAPGGGN. The elastin domain is preferably represented by the following sequences: VPGXG (where X = V, I, A) or JGGZG (where J, Z = V, L, A), and most preferably VPGIG and VPGAG. The silk fibroin domain is preferably represented by the following sequence: GAGAGS. The recombinant hybrid protein of the present invention is rich in functional domains, preferably:

[0091] - RGD motif of fibronectin cell adhesion sequence; most preferably AVTGRGDSPASS, and optionally shortened GRGDSP or extended TVY AVTGRGDSPASS;

[0092] - MMP motifs recognized by matrix metalloproteinases, with GPQGIWGQ being the most preferred;

[0093] - The lysine-rich K+ cross-linking domain can chemically modify recombinant structural proteins; the optimal choice is GGKGGKGGGKGG.

[0094] -ZIP domain encoding the leucine zipper-stabilized supramolecular structure; optimally selected: VGGGGGKENQIAIRASFLEKENSALRQEVADLRKELGKCKNILAKYEAGGGGG.

[0095] Without departing from the scope of this invention, those skilled in the art can make quantitative or qualitative changes to the functional domains to adjust the properties of the recombinant hybrid protein of this invention.

[0096] Recombinant hybrid protein sequences enriched with RGD domains derived from fibronectin and integrin-binding sequences promote the adhesion of various endothelial cells, smooth muscle cells, and fibroblasts; therefore, biomaterials rich in recombinant hybrid proteins have a positive effect on the growth of cells in physical contact with them. Metalloproteinase-sensitive MMP sequences derived from human α(I) collagen chains are added to the recombinant structural proteins to promote protein degradation associated with extracellular matrix rearrangement that may be induced by proliferating cells. The presence of K+ motifs aims to functionalize peptides with selected chemical groups, thereby promoting controlled cross-linking of biomaterials rich in recombinant hybrid proteins, which is required for bioprinting. Recombinant structural proteins enriched with ZIP sequences derived from the dimerization domain of hepatitis leukemia factor (HLF) are expected to provide structural stability during the printing process due to their ability to form amphiphilic α-helical structures based on hydrophobic interactions.

[0097] Utilizing the domain combination described in this invention, the developed protein can be applied to polymeric biomaterials for cell remodeling, and due to its universal importance as a collagenase substrate, it can also mediate cell invasion, migration, proliferation, and the growth of new tissues. The resulting recombinant hybrid protein will be able to modulate cell growth by fine-tuning the RGD density and introducing MMP-sensitive domains into the constructed hydrogel, thereby mimicking the extracellular matrix (ECM).

[0098] Example

[0099] Example 1

[0100] The recombinant RE15mR heterozygous protein consists of the following sequences: 3 arthropod elastin domains, 1 K+ domain, 3 arthropod elastin domains, 1 MMP domain, 3 elastin domains, 1 K+ domain, 4 elastin domains, 1 K+ domain, 3 elastin domains, 1 K+ domain, 4 elastin domains, 1 K+ domain, 3 elastin domains, 1 MMP domain, and 7 RGD domains.

[0101] Figure 1 A schematic diagram of the RE15mR recombinant hybrid protein is shown. Figure 4 The DNA sequence of SEQ #1 encoding the RE15mR recombinant heterozygous protein is shown. Figure 6 The amino acid sequence of the RE15mR recombinant heterozygous protein is shown in SEQ #3.

[0102] According to a preferred embodiment, the amino acid sequence of the RE15mR recombinant heterozygous protein contains 12-15% proline and 32-38% glycine, and the molecular weight of the protein is approximately 26 kDa.

[0103] Example 2

[0104] The recombinant hybrid protein EJ17zipR consists of one ZIP domain, 56 elastin domains, 10 silk fibroin domains, and 7 RGD domains. Figure 2 A schematic diagram of the recombinant EJ17zipR heterozygous protein is shown. Figure 5 The DNA sequence encoding the recombinant heterozygous protein EJ17zipR is shown in SEQ #2. Figure 7 The amino acid sequence of the recombinant heterozygous protein EJ17zipR is shown in SEQ #4.

[0105] According to a preferred embodiment, the amino acid sequence of the recombinant hybrid protein EJ17zipR contains 12-17% proline and 20-30% glycine, and the molecular weight of the protein is approximately 86 kDa.

[0106] Proteins should oligomerize into molecules larger than 1 mDa (verification method - polyacrylamide gel electrophoresis under native conditions).

[0107] Example 3

[0108] To obtain the recombinant heterozygous protein of the present invention, a plasmid vector containing a promoter derived from bacteriophage T7, namely pET11a, was used. Using methods known in the field of molecular cloning techniques, and employing selective restriction sites, the DNA sequence encoding the recombinant heterozygous protein of the present invention, namely the DNA sequence encoding the recombinant heterozygous protein RE15mR, SEQ #1 (…), was… Figure 4 ) or the DNA sequence encoding the recombinant heterozygous protein EJ17zipR SEQ#2 ( Figure 5 It is integrated into the carrier.

[0109] Specifically, the restriction sites are Ndel and BamHI. Using sites with such... Figure 3 The cloned sequence was amplified using selected primers based on the given sequence. An expression vector capable of efficiently and stably expressing the recombinant protein in E. coli cells from strain BLR(DE3) was obtained.

[0110] Figure 8 A map of the expression vector containing the cloned gene encoding the RE15mR heterozygous protein is shown. Figure 9 A map of the expression vector carrying the cloned gene encoding the EJ17zipR heterozygous protein is shown.

[0111] Example 4

[0112] The recombinant hybrid proteins RE15mR and EJ17zipR were obtained by a method comprising the following steps:

[0113] 1. Escherichia coli cell cultures transformed with a plasmid encoding a recombinant heterozygous protein from a BLR(DE3) strain [optionally BL21(DE3)], supplemented with an appropriate antibiotic (preferably 50-200 μg / ml ampicillin). This method uses conventional culture media, selected according to the host strain used. For the BL21(DE3) and BLR(DE3) strains used, the culture medium can be standard nutrient-rich LB medium with additional proline and glycine. Cultures in the bioreactor use inorganic salt media, the composition of which was initially developed experimentally during the development of this invention. The composition of the culture media used is shown in Table 1 below:

[0114] Table 1: Culture medium composition

[0115]

[0116] Growth cultures using this medium, especially large-scale cultures in bioreactors, offer the advantage of relatively low cost while ensuring satisfactory growth levels (comparable to those of media used in laboratory-scale cultures). Bacterial biomass is produced in the first stage of cultivation, with the following parameters: temperature: 30°C, stirring: 150-700 rpm, aeration rate: 5-10 LPM (liters / minute), DO (dissolved oxygen): >20%, pH: 7.1 + / -1. Cultivation continues until an OD600 optical density of 0.7-0.9 is achieved. Next, the expression of recombinant heterozygous proteins was induced by adding isopropyl-β-D-1-thiogalactoside (IPTG) to a concentration of 0.4–1.0 mM or by adding lactose to a concentration of 5–20 mM, and cultured under the following conditions: temperature: 37 °C, stirring: 500–700 rpm, aeration: 7–10 LPM, DO: > 20%, pH: 7.1 + / -1, for 5–7 hours until an OD600 optical density of 3.0–9.0 was reached.

[0117] 2. In this stage, bacterial biomass is separated from the culture medium by centrifugation. The bacterial cells are then suspended in a lysis buffer prepared for this purpose, as shown in Table 2 below.

[0118] Table 2: Composition of Lysis Buffer

[0119]

[0120] Bacterial biomass was disrupted using a high-pressure flow breaker at a pressure of 800-900 bar. During operation, 3 to 4 rounds of disruption were preferred, and 0.4% (w / v) polyethyleneimine (PEI) was added to the suspension to precipitate host DNA.

[0121] 3. Insoluble protein fractions, undissociated bacterial cells, and precipitated DNA were separated from the supernatant containing recombinant heterozygous proteins by centrifugation.

[0122] 4. Incubate the supernatant containing the recombinant heterozygous protein at 85-95°C for 15-30 minutes, then centrifuge to remove the denatured protein. Experiments conducted also showed that adding a protease inhibitor mixture (preferably complete, EDTA-free, Roche, product number: 05056489001) to the recombinant protein solution at this stage, at a dosage of 1 tablet per 50 mL of solution.

[0123] 5. At room temperature, precipitate the recombinant protein from the solution by adding ammonium sulfate to 10% saturation (for EJ17zipR protein) or 20-40% saturation (for RE15mR protein), then centrifuging the precipitated protein and dissolving it in 20-40 mM TRIS buffer (containing 10 mM EDTA pH 8.0).

[0124] 6. Dialyze the recombinant protein suspension in 20-40 mM TRIS buffer (containing 10 mM EDTA, pH 8.0) at 4°C for 24-48 hours.

[0125] 7. Protein purification was performed using Macro-Prep High Q media (Bio-Rad). The resin-packed column was equilibrated with a calibration buffer consisting of 20–50 mM TRIS buffer (pH 8.0). The protein solution obtained after dissolving the salted-out protein was applied to the equilibrated column. Separation was performed using rapid protein liquid chromatography (FPLC). Proteins not bound to the matrix were eluted with the calibration buffer. Proteins bound to the resin were eluted with an elution buffer consisting of 20–50 mM TRIS buffer (pH 8.0) + 1 M NaCl. Fractions with absorbance higher than 0.05 AU (absorbance units) were collected at a flow rate of 1–2 mL / min. The concentration of eluted proteins was determined using the Bradford and BCA method (Pierce BCA Protein Assay Kit). The recombinant hybrid proteins RE15mR and EJ17zipR of this invention are not bound to the resin but are eluted from the column with the calibration buffer.

[0126] 8. Remove endotoxins, preferably using Pierce high-capacity endotoxin removal resin columns according to the manufacturer's instructions.

[0127] 9. Dialyze the recombinant protein suspension in ddH2O at 4°C for 24-48 hours.

[0128] 10. Lyophilized recombinant hybrid protein.

[0129] The method described above is characterized in that the obtained recombinant RE15mR protein can form a gel at 4°C, a concentration higher than 200 mg / mL, and with the addition of 0.5M NaCl. The recombinant EJ17zipR protein does not form a hydrogel. The yield of this method is 20-70 mg of recombinant RE15mR and EJ17zipR protein per liter of culture. Figure 10 shows the purification process of the recombinant RE15mR structural protein under denaturing conditions via SDS-PAGE electrophoresis, comparing impurities. This figure shows the SDS-PAGE separation images of samples collected from subsequent stages of obtaining the recombinant hybrid protein RE15mR, where the numbers in Figure A represent: 1- *E. coli* BLR(DE3) culture before induction; 2- *E. coli* BLR(DE3) culture after IPTG induction; 3- LMW protein molecular weight (mass) standard; 4- supernatant after sonication; 5- precipitate after sonication; 6- sonicated and then... 7- Supernatant after incubation at 0℃; 8- Precipitate after sonication and incubation at 90℃; 9- Supernatant after sonication, incubation, and precipitation with 0-20% saturated ammonium sulfate; 10- Supernatant after sonication, incubation, and precipitation with 20-40% saturated ammonium sulfate; 11- Precipitate after sonication, incubation, and precipitation with 20-40% saturated ammonium sulfate; 12- Supernatant after dialysis I; 13- Precipitate after dialysis I. The numbers in Figure B represent: 1- LMW protein molecular weight standard; 2- Macro-Prep 3 - Fractions unrelated to Macro-Prep High Q Media resin; 4 - Fractions bound to Macro-Prep High Q Media resin; 5 - Fractions entering the Pierce high-volume endotoxin removal resin column; 6 - Fractions eluted from the high-volume endotoxin removal resin; 7, 8, 9, 10 - Final protein solution for lyophilization.

[0130] Figure 11 shows the purification process of the recombinant structural protein EJ17zipR under denaturing conditions via SDS-PAGE electrophoresis, comparing impurities. The figure displays SDS-PAGE separation images of samples collected in subsequent stages after obtaining the recombinant EJ17zipR heterozygous protein. The numbers in Figure A represent: 1- *E. coli* BLR(DE3) culture before induction; 2- *E. coli* BLR(DE3) culture after induction; 3- Broad Multicolor (BMW)... (Color) Protein molecular weight standard; 4- Supernatant after sonication; 5- Precipitate after sonication; 6- Supernatant after sonication and incubation at 90°C; 7- Precipitate after sonication and incubation at 90°C; 8- Supernatant after sonication, incubation, and salting out in 0-10% saturated ammonium sulfate; 9- Precipitate after sonication, incubation, and salting out in 0-10% saturated ammonium sulfate; 10- Supernatant after dialysis I; 11- Precipitate after dialysis I. The numbers in Figure B represent: 1- LMW protein molecular weight standard; 2- Sample loading onto Macro-Prep High Q Media resin; 3- Fraction unrelated to Macro-Prep High Q Media resin; 4- Fraction bound to Macro-Prep High Q Media resin; 5- Fraction entering Pierce high-volume endotoxin removal resin; 6- Fraction eluted from high-volume endotoxin removal resin; 7, 8, 9, 10- Final protein solution for lyophilization.

[0131] Example 5

[0132] The recombinant hybrid proteins RE15mR and EJ17zipR of the present invention are subjected to methacrylation. In a preferred embodiment, the method includes the following steps:

[0133] 1. Weigh the batch of the recombinant hybrid protein of the present invention to be subjected to methacrylation treatment into a reaction vessel with a capacity of X mL using an analytical balance.

[0134] Note: All mass values ​​should be retained to 3 significant figures. The choice of reaction vessel capacity (X mL) depends on the volume of the final reaction mixture, which should not exceed 60% of the total vessel capacity.

[0135] 2. The reaction vessel is equipped with a mixing element and placed above a magnetic stirrer. Using an automated pipette, disperse X mL of PBS x 1 buffer into the vessel to obtain a 2% (w / v) solution. The stirrer speed is set in the range of 200 to 1500 rpm, and in a preferred embodiment, approximately 1000 rpm. Continue stirring the mixture at room temperature until the substrate is completely dissolved (typically about 10 minutes).

[0136] Note: In addition to PBSx1, any buffer that can maintain the pH at neutral or above the isoelectric point of the protein can be used.

[0137] 3. After the substrate is completely dissolved, place the reaction vessel in an ice-water bath (fill the crystallizer with water and ice to half its volume to maintain the instability of the liquid phase) and protect it from light (e.g., wrap the entire container with aluminum foil). The expected bath temperature is ≤4°C.

[0138] 4. Use an automated pipette to measure X mL of methacrylic anhydride (MMA) or other electrophilic reagent and add it to the reaction.

[0139] Note: The key parameter describing the efficiency of the methacrylation reaction is the degree of substitution (DS), which is controlled by adjusting the amount of methacrylic anhydride (MMA) used in the reaction.

[0140] In protein methacrylation reactions, the appropriate amount of methacrylic anhydride (or other nucleophilic reagent) must be selected each time based on the given protein sequence. This requires reading the amino acid sequence of the protein and determining the number of lysine residues in the protein chain. Then, considering the total mass of 1 mole of protein and the mass weighed into the reaction, the ratio of free lysine amino groups to the anhydride is calculated. The amount of methacrylic anhydride used. The following formula should be used for calculations – if the nucleophile is in a liquid state:

[0141]

[0142] in:

[0143]

[0144] Alternatively, if the nucleophile is solid, then according to the following formula:

[0145]

[0146] in:

[0147]

[0148] The amount of methacrylic anhydride used is a parameter for controlling the degree of substitution. Depending on the expected degree of methacrylation, an anhydride in excess of 1 to 10 times the free amino group of lysine can be used. Therefore, the volume of the anhydride calculated according to the above formula should be multiplied by a value in the range of 1 to 10.

[0149] Besides the amino group of lysine, other amino acid units, such as tryptophan, tyrosine, lysine, threonine, serine, or arginine, can also be methacrylated, provided they are present in the amino acid sequence. These factors must be taken into account when calculating the degree of protein substitution. Based on the calculations, to methacrylate a single protein (considering a 1:1 ratio of methacrylic anhydride to the lysine amino group), the following substances should be added dropwise:

[0150]

[0151] 5. The reaction is carried out under given conditions (T≤4°C, stirring at 200 to 1500 rpm, approximately 1000 rpm in the preferred embodiment) until the reaction is complete, preferably 24 h.

[0152] 6. After the required reaction time, add the PBSx1 solution in portions to the mixture until the mixture is diluted 5 times (1:4 mixture:PBSx1).

[0153] 7. Pour the resulting solution into a dialysis tube following the steps below.

[0154] a) Measure and cut the dialysis tubing to the appropriate length.

[0155] b) Place the tube in a beaker containing deionized water and let it stand for 10 minutes. Then replace the water with clean water and let it stand for 10 minutes. Finally, soak it in a 40% ethanol-water solution for 10 minutes.

[0156] c) Gently remove the soaked tube from the beaker and tie one end tightly. Place a funnel over its open end and pour in a small amount of water to check the tube's tightness.

[0157] d) If there is no leakage, remove the water from the tube and then quantitatively transfer the solution into it, rinsing the reaction vessel with 3 x 1 mL of deionized water.

[0158] e) Tighten the tube and secure it with clamps on both sides, then place it in a 5 L beaker.

[0159] f) Fill a beaker with deionized water (about 4 L) and place it in the refrigerator.

[0160] 8. Perform dialysis at 4℃ for 2 days, changing the water once a day.

[0161] 9. After the dialysis process is complete, quantitatively transfer the solution in the dialysis tube to a large beaker, and then about 5 mL (using an automatic pipette) to a small glass vial.

[0162] Note: Each empty glass vial should be weighed and its mass recorded as m1. Then pour the solution into it, weigh it again, and record the mass as m2.

[0163] 10. Freeze the solution vials at -80°C, preferably for at least 3 hours. Then freeze-dry according to the following parameters:

[0164] a) Shelf temperature: 0°C

[0165] b) Pressure: 0.100 mbar

[0166] c) Duration: 48h.

[0167] 11. Weigh the vials containing the obtained lyophilized material and record the mass as m3. Then, calculate Δm = m3 - m1 to determine the mass of each vial of lyophilized material.

[0168] 12. Store the material vials at -20°C.

[0169] 13. Use 1H NMR spectroscopy to analyze the degree of substitution of the final product.

[0170] use 1 Results of H NMR spectroscopy analysis on the degree of substitution of methacrylic acid groups in RE15mR protein

[0171] The approximate degree of substitution of the RE15mR protein was determined. Figure 12A This shows the protein before the methacrylation reaction. 1 The 1H NMR spectrum showed no signal observed in the chemical shift region of 5 ppm–6.5 ppm. Figure 12B The protein after methacrylation was shown. 1¹H NMR spectra were used, with methacrylic anhydride used for methacrylation in a 2-fold excess relative to the lysine amino group. The degree of substitution was determined by comparing the signal integral of the tryptophan aromatic ring at 7.47 ppm. The signal integral was 0.09, equivalent to 2 amino acids per mole in the protein chain. The integral of the methacrylic acid group attached to tryptophan was also 0.09, indicating that tryptophan was 100% substituted. Considering that there are 6 tyrosine, 7 lysine, 7 threonine, 31 serine, and 13 arginine units per mole in the protein chain, the signal of the methacrylic acid group attached to a single amino acid was integrated and compared with the tryptophan signal, and the degree of substitution (DS) of the methacrylic acid group was calculated based on this. The spectra showed that the methacrylic acid groups attached to tryptophan (6.34 ppm and 5.90 ppm) had a DS of ~100%, tyrosine (5.66 ppm and 5.42 ppm) had a DS of ~100%, and lysine (5.81 ppm and 5.53 ppm) had a DS of ~100%. Furthermore, overlapping signals of protons of methacrylic acid groups linked to threonine, serine, and arginine were observed in the 5.68–5.76 ppm and 5.44–5.50 ppm regions. However, the degree of substitution of individual amino acids could not be definitively determined due to signal overlap and low spectral resolution caused by the low functionalization of the remaining amino acids, as well as the influence related to protein molecule size.

[0172] Figure 12C The methacrylated RE15mR protein (from batch 15_E_003) was shown. 1 ¹H NMR spectra, in which methacrylic anhydride was used for methacrylation in 10-fold excess relative to the lysine amino group. The spectra show that the methacrylic acid group is attached to the following amino acids: tryptophan (6.34 ppm and 6.01 ppm), tyrosine (5.66 ppm and 5.41 ppm), lysine (5.81 ppm and 5.53 ppm), threonine (5.90 ppm and 5.68 ppm), serine (5.64 ppm and 5.31 ppm), and arginine (6.11 ppm and 5.73 ppm).

[0173] use 1 Results of H NMR spectroscopy analysis of the degree of substitution of methacrylic acid groups in EJ17zipR protein

[0174] Figure 12D This shows the protein before the methacrylation process. 1 The 1H NMR spectrum showed no signal observed in the 5 ppm–6.5 ppm region. Figure 12E The protein after methacrylation was shown. 1The 1H NMR spectrum, in which methacrylic anhydride was used for methacrylation in 2-fold excess relative to the lysine amino group, shows the presence of methacrylic acid groups, along with signals from protons originating from the double bond (shifts 5.73 ppm and 5.39 ppm) and from the methyl group (shift 1.18 ppm). Signals from aromatic protons of phenylalanine located in the protein chain are observed at 7.27 ppm and 7.21 ppm. The integral of this signal (2.48) corresponds to 145 aromatic protons located in a ring of 29 phenylalanine units per mole in the protein structure. The signal integral for the methacrylic acid protons is 0.12. Considering the number of protons from phenylalanine, it is determined that there are 6 methacrylic acid groups attached to the protein chain per mole of protein. However, due to the complexity of the resulting spectrum and its low resolution in the allyl region, it is not possible to clearly determine which amino acid the methacrylic acid group is attached to. Therefore, the exact degree of substitution at the functionalization site cannot be determined. However, due to the presence of chemical shifts, we assume that the results correspond to 100% substitution of the lysine amino group.

[0175] Example 6

[0176] The bioactivity of the recombinant hybrid protein of this invention was tested, including its effects on cell viability, proliferation, adhesion, and cytotoxicity. Protocols, reagents, and equipment commonly used in such studies were employed. The control in this study was fibronectin, coated at a concentration of 1 μg / cm². The negative control consisted of wells coated with no protein.

[0177] Composition of culture medium

[0178] DMEM (Duborough Modified Eagle Medium) is supplemented with 10% FBS, 4 mM L-glutamine, 4.5 g / L glucose, 1 mM sodium pyruvate, 1500 mg / L sodium carbonate, and 50 IU / ml penicillin and 50 μg / ml streptomycin.

[0179] Ready-made culture media: Endothelial cell growth kit - VEGF, ATCC primary cell solution, catalog number PCS-100-041

[0180] Duchenne Modified Eagle Medium - High Glucose, Sigma, D6429

[0181] Cell lines used in the test

[0182] L929-mouse fibroblasts, monolayer adherent cells, ATCC catalog number CCL-1

[0183] Human umbilical vein endothelial cells (HUVECs), passage II, ATCC catalog number PCS-100-010

[0184] Human dermal fibroblasts HDFα, ATCC catalog number PCS-201-012

[0185] AlamarBlue staining reagent

[0186] AlamarBlue™ Cell Viability Reagent (Invitrogen, catalog number DALI 100); the active ingredient, resazurin, is a non-toxic blue compound. Upon entering living cells under reducing conditions, resazurin is induced to convert into resorufin, which emits strong red fluorescence. This indicates the number of metabolically active living cells.

[0187] Use commercially available LIVE / DEAD TM The kit (Invitrogen, catalog number L3224) is used for mammalian cell viability / cytotoxicity testing.

[0188] Calcein AM and EthD-1 staining

[0189] Cell viability assessment – ​​calcein derivative staining. Non-fluorescent acetylmethoxycalcein (calcein AM) can freely penetrate the cell membrane and is converted into strongly fluorescent green calcein by intracellular esterases.

[0190] EthD-1 ethidium homodimer can pass through damaged membranes into cells and, upon binding to nucleic acids, exhibits a forty-fold increase in bright red fluorescence, thus enabling the detection of dead cells in a colony.

[0191] Calcein AM / EthD-1 staining not only allows for qualitative and quantitative assessment of the ratio of live to dead cells in a population, but also preserves their shape and position. Unlike the MTT method (the reference method using formazan staining), it does not require cell disintegration for measurement (absorbance / fluorescence) and allows for in situ microscopic observation.

[0192] 6.1 Bioactivity of RE15mR protein

[0193] Effects of RE15mR protein on cell proliferation

[0194] RE15mR protein obtained from Escherichia coli DE3 BLR strain culture was purified and lyophilized for coating 96-well plates for suspension culture of eukaryotic cells (unmodified surface). The RE15mR protein aqueous solution was prepared at 1 and 5 μg / cm³. 2 The appropriate amount was applied to the culture plate and dried. The control in the study was 1 μg / cm². 2The amount used was for coating fibronectin. The negative control was the uncoated wells. L929 or HUVEC cell lines were loaded at 5 x 10⁻⁶ cells / well. 3 Cells were seeded in wells onto prepared plates. Cells were incubated in dedicated culture medium for 2, 24, and 48 hours. Afterward, the wells were washed with sterile phosphate-buffered saline (PBS) to remove dead and non-adherent cells. Cells were stained with Alamar Blue reagent for 3 hours, and fluorescence was measured at 530 nm (excitation) and 590 nm (emission) wavelengths.

[0195] Figure 13A The fluorescence level of AlamarBlue staining of L929 cells on RE15mR protein-coated plates compared to that on fibronectin-coated plates is shown in the L929 cell proliferation assay. Figure 13B Similar results were shown from studies using HUVEC.

[0196] Considering the division time of the L929 cell line, the fluorescence intensity increased after 48 hours compared to 2 and 24 hours. Figure 13A This indicates that active cell proliferation was present in both the positive control and the wells coated with the test protein. Therefore, it can be concluded that the RE15mR protein does not interfere with the cell proliferation rate used in cell line studies. The same observation also applies to HUVEC studies. Figure 13B (but only up to 1 μg / cm) 2 The blanket.

[0197] The effect of RE15mR protein on cell adhesion

[0198] RE15mR protein obtained from Escherichia coli DE3 BLR strain culture was purified and lyophilized for coating 96-well plates for suspension culture of eukaryotic cells (unmodified surface). The RE15mR protein aqueous solution was prepared at 1 and 5 μg / cm³. 2 The appropriate amount was applied to the culture plate and dried. The control in the study was 1 μg / cm². 2 The amount used was for coating fibronectin. The negative control showed no protein coating in the wells. L929 or HUVEC cell lines were loaded at 1x10⁻⁶ cells / well. 4 Cells were seeded in wells onto prepared plates. Cells were incubated in dedicated culture medium for 2, 4, and 24 hours. Afterward, the wells were washed with sterile phosphate-buffered saline (PBS) to remove dead and non-adherent cells. In this example, washing was to remove non-adherent cells. Cells were stained with Alamar Blue reagent for 3 hours, and fluorescence was measured at 530 nm (excitation) and 590 nm (emission) wavelengths.

[0199] Figure 14AThe fluorescence level of L929 cell cultures on RE15mR protein-coated plates compared to fibronectin-coated plates was shown in the L929 cell proliferation assay. Figure 14B Similar results were shown from studies using HUVEC.

[0200] Increase in fluorescence during incubation ( Figure 14A This indicates that cells adhered to the plastic used, both in the positive control and in the wells coated with the test protein. The RE15mR protein was observed to promote cell adhesion to a degree comparable to fibronectin—a commercially available protein used to coat surfaces used for eukaryotic cell cultures—in cell line studies. Similar observations were also observed in the HUVEC study. Figure 14B (), but only for 1 μg / cm 2 The blanket.

[0201] Cytotoxicity of RE15mR protein on L929 cells

[0202] RE15mR protein obtained from Escherichia coli DE3 BLR strain culture was purified and lyophilized, then used to coat 96-well plates for suspension culture of eukaryotic cells (unmodified surface). The RE15mR protein aqueous solution was added at 1 μg / cm³. 2 The amount was applied to the culture plate and dried. The control in the study was also applied at 1 μg / cm. 2 The amount used was for coating fibronectin. L929 cell lines were loaded at 2 x 10⁻⁶. 4 / hole, 1x10 4 / hole and 5x10 3 Cells were seeded in wells at a rate of / wells onto prepared plates. Cells were incubated in dedicated culture medium for 2, 24, 48, and 72 hours. The medium was then removed from the selected wells, cells were washed with 70% methanol and incubated for 30 minutes, with a 100% dead cell population as a negative control. Cells were stained with calcein AM (1 μM) and ethidium homodimer (EthD1, 2 μM) for 30 minutes, and fluorescence was measured at 485 and 530 nm (excitation) and 530 and 645 nm (emission) wavelengths.

[0203] Figure 15 The fluorescence levels of L929 cell cultures on RE15mR-coated plates versus fibronectin-coated plates are shown in the RE15mR protein cytotoxicity assay. This is based on the increase in fluorescence during incubation. Figure 15 It can be concluded that, regardless of the culture time, the RE15mR protein used to coat the culture plastic is less cytotoxic to the L929 cell line than fibronectin, which is commonly used for this purpose.

[0204] Cytotoxicity of blends containing RE15mR protein against HUVEC cells

[0205] The cytotoxicity of the blend was tested using a "contact" method, in which HUVEC cells were mixed with HDFα at a ratio of 1:2 and seeded into wells covered with a cross-linked material containing the recombinant blend protein.

[0206] After lyophilization, a blend was prepared using RE15mR protein, based on a 10% methacrylamide gelatin solution containing 0.5 or 0.1 mg / ml of the detection protein.

[0207] In the contact assay, the resulting blend was coated onto 48-well plates used for culturing eukaryotic cells. The cell line mixture was then cultured at 2.5 x 10⁻⁶ cells / well. 5 / hole, 1.25x10 5 / hole and 0.625x10 5 The total number of cells was seeded into the prepared plates. The cells were incubated in a dedicated culture medium for 24, 48, and 96 hours.

[0208] Cells were stained with calcein AM (1 μM) and ethidium homodimer (EthD1, 2 μM) for 30 minutes. Green (live cells) and red (dead cell nuclei) fluorescence were observed using an Olympus 1X83 microscope equipped with CellSens software for photographic recording. Figure 16 A summary of photographs of the various blends is shown, displaying the fluorescence of stained cells at subsequent time points. The control group consisted of cells seeded on TC-modified plastic (suitable for adherent culture) and GelMa blends, and also included photographs of cultures on surfaces containing 0.5 mg / ml RE15mR protein (B15H) and 0.1 mg / ml RE15mR (B15L). The photographs were taken at 4x and 10x magnification, respectively.

[0209] In contact experiments on HUVEC and HDFα cells grown on a blend supplemented with RE15mR protein, no cytotoxic effects of the test protein were observed at either test concentration. Cells were in an active dividing state, with negligible dead cells that were directly proportional to the total number of growing cells.

[0210] Absorbance measurement in the MTT assay for RE15mR protein cytotoxicity against L929 cells.

[0211] The cytotoxicity of RE15mR protein to L-929 cells was tested according to ISO 10993-5:2009(E): Biological evaluation of medical devices – Part 5: In vitro cytotoxicity studies (absorbance measurement in MTT assay). Cells were cultured at 1 x 10⁻⁶ cells according to the planned exposure time. 5 / ml and 5x10 4 The culture was seeded at a density of 100 μl / ml into a 96-well plate.

[0212] Under standard conditions (5% CO2 and 37°C), cells were cultured overnight in supplemented DMEM medium to allow fibroblasts to diffuse to the bottom of the culture vessel. The assay employed a direct method, adding a purified protein solution reconstituted in the medium to the culture. After examining confluence and colony status, 100 μl of the protein solution from the medium was placed on plates at concentrations of 1, 0.5, and 0.1 mg / mL. Cell densities were set at 1 x 10⁻⁶ cells / mL. 5 Incubate at 24 hours with a plate temperature of 5 x 10⁶ ml. 4 / ml of culture was exposed to RE15mR protein for 48 hours.

[0213] Subsequently, the cells were incubated with MTT reagent solution for 2 hours, then all liquid above the cells was removed, and the formed formazan crystals were dissolved in DMSO. The amount of colored product formed was determined to be proportional to the number of viable cells by measuring absorbance at 570 and 650 nm wavelengths. According to standards, the expected result is that the cell viability exposed to the cytotoxic agent is not less than 70% of the viability of untreated cells in the negative control.

[0214] Based on the results, it was concluded that RE15mR protein showed no cytotoxicity to the L-929 fibroblast cell line at 24 and 48 hours of exposure within the tested concentration range (0.1 - 1 mg / ml). Figure 17 ).

[0215] 6.2 Bioactivity of EJ17zipR protein

[0216] Effects of EJ17zipR protein on cell proliferation

[0217] EJ17zipR protein obtained from Escherichia coli DE3 BLR strain culture was purified and lyophilized for coating 96-well plates for suspension culture of eukaryotic cells (unmodified surface). The EJ17zipR protein aqueous solution was prepared at 1 and 5 μg / cm³. 2 The appropriate amount was applied to the culture plate and dried. The control in the study was 1 μg / cm². 2 The amount used was for coating fibronectin. The negative control showed no protein coating in the wells. L929 or HUVEC cell lines were loaded at 5 x 10⁻⁶ cells / well.3 Cells were seeded in wells onto prepared plates. Cells were incubated in dedicated culture medium for 2, 24, and 48 hours. Afterward, the wells were washed with sterile phosphate-buffered saline (PBS) to remove dead and non-adherent cells. Cells were stained with Alamar Blue reagent for 3 hours, and fluorescence was measured at 530 nm (excitation) and 590 nm (emission) wavelengths.

[0218] Figure 18A The fluorescence level of AlamarBlue staining of L929 cells on EJ17zipR protein-coated plates versus fibronectin-coated plates is shown in the L929 cell proliferation assay. Figure 18B Similar results were shown from studies using HUVEC.

[0219] Considering the division time of the L929 cell line, the fluorescence intensity was enhanced after 48 hours compared to 2 and 24 hours. Figure 18A This indicates that active cell proliferation was present in both the positive control and the wells coated with the test protein. The slightly lower signal obtained from the test protein may be due to the lower cell adhesion to EJ17zipR protein compared to fibronectin. Nevertheless, it can still be concluded that EJ17zipR protein does not interfere with the cell proliferation rate used in cell line studies. The same observation also applies to HUVEC studies. Figure 18B (), but only for 1 μg / cm 2 The blanket.

[0220] Effects of EJ17zipR protein on cell adhesion

[0221] EJ17zipR protein obtained from Escherichia coli DE3 BLR strain culture was purified and lyophilized for coating 96-well plates for suspension culture of eukaryotic cells (unmodified surface). The EJ17zipR protein aqueous solution was prepared at 1 and 5 μg / cm³. 2 The appropriate amount was applied to the culture plate and dried. The control in the study was 1 μg / cm². 2 The amount used was for coating fibronectin. The negative control showed no protein coating in the wells. L929 or HUVEC cell lines were loaded at 1x10⁻⁶ cells / well. 4 Cells were seeded into prepared plates at a concentration of / wells. Cells were incubated in dedicated culture medium for 2, 4, and 24 hours. Afterward, the wells were washed with sterile phosphate-buffered saline (PBS) to remove dead and non-adherent cells. In this example, the purpose of washing was to remove non-adherent cells. Cells were stained with Alamar Blue reagent for 3 hours, and fluorescence was measured at 530 nm (excitation) and 590 nm (emission) wavelengths.

[0222] Figure 19AThe fluorescence level of L929 cell cultures on EJ17zipR protein-coated plates compared to fibronectin-coated plates was shown in the L929 cell adhesion assay. Figure 19B Similar results were shown in studies using HUVEC cells.

[0223] Increase in fluorescence during incubation ( Figure 19A This indicates that cells adhered to the plastic used, both in the positive control and in wells coated with the test protein. The EJ17zipR protein was observed to promote cell adhesion used in cell line studies, although the number of adherent cells was lower compared to fibronectin. The effect of protein concentration on cell adhesion was clearly visible—the number of adherent cells increased after only 24 hours of culture with increasing protein concentration. The same observations also applied to the HUVEC studies. Figure 18B (), but only for 1 μg / cm 2 The blanket.

[0224] Cytotoxicity of EJ17zipR protein on L929 cells

[0225] EJ17zipR protein obtained from Escherichia coli DE3 BLR strain culture was purified and lyophilized, then used to coat 96-well plates for suspension culture of eukaryotic cells (unmodified surface). The EJ17zipR protein aqueous solution was added at 1 μg / cm³. 2 The amount was applied to the culture plate and dried. The control in the study was also applied at 1 μg / cm. 2 The amount used was for coating fibronectin. L929 cell lines were loaded at 2 x 10⁻⁶. 4 / hole, 1x10 4 / hole and 5x10 3 Cells were seeded in wells at a rate of / wells onto prepared plates. Cells were incubated in dedicated culture medium for 2, 24, 48, and 72 hours. The medium was then removed from the selected wells, and the cells were washed with 70% methanol and incubated for 30 minutes to obtain a negative control with a 100% dead cell population. Cells were stained with calcein AM (1 μM) and ethidium homodimer (EthD1, 2 μM) for 30 minutes, and fluorescence was measured at excitation wavelengths of 485 and 530 nm and emission wavelengths of 530 and 645 nm.

[0226] Figure 20 The fluorescence level of L929 cell cultures on EJ17zipR protein-coated plates compared to fibronectin-coated plates is shown in the cytotoxicity assay of L929 cells. This is based on the increase in fluorescence during incubation. Figure 20We can conclude that the EJ17zipR protein used to coat culture plastics is not cytotoxic to L929 cells, but compared with fibronectin, which is usually used for this purpose, we observed a significantly lower fluorescence signal after 72 hours of culture and propagation.

[0227] Cytotoxicity of cells containing EJ17zipR protein blend

[0228] The cytotoxicity of the blend was tested using a "contact" method, in which HUVEC cells were mixed with HDFα at a ratio of 1:2 and seeded into wells covered with the cross-linked blend.

[0229] After lyophilization, a blend was prepared using EJ17zipR protein, based on a 10% methacrylamide gelatin solution containing 0.5 or 0.1 mg / ml of the test protein.

[0230] In the contact assay, the resulting blend was coated onto 48-well plates used for culturing eukaryotic cells. The cell line mixture was then cultured at 2.5 x 10⁻⁶ cells / well. 5 / hole, 1.25x10 5 / hole and 0.625x10 5 The total number of cells was seeded into the prepared plates. The cells were incubated in a dedicated culture medium for 24, 48, and 96 hours.

[0231] Cells were stained with calcein AM (1 μM) and ethidium homodimer (EthD1, 2 μM) for 30 minutes. Green (live cells) and red (dead cell nuclei) fluorescence were observed using an Olympus 1X83 microscope equipped with CellSens software for photographic recording. Figure 21 A compilation of photographs of the various blends is shown, displaying the fluorescence of stained cells at subsequent time points. The control group consisted of cells seeded on TC-modified plastic (suitable for adherent culture) and GelMa blends. Also included were photographs of cultures on surfaces containing 0.5 mg / ml EJ17zipR protein (B17H) and 0.1 mg / ml EJ17zipR (B17L). The photographs were taken at 4x and 10x magnification, respectively.

[0232] In contact experiments on HUVEC and HDFα cells grown on a blend supplemented with EJ17zipR protein, no cytotoxic effects of the test protein were observed at either test concentration. Cells were in an active dividing state, with negligible dead cells that were directly proportional to the total number of growing cells.

[0233] Absorbance measurement in the cytotoxicity-MTT assay of EJ17zipR protein against L929 cells.

[0234] The cytotoxicity of EJ17zipR protein to L-929 cells was tested according to ISO 10993-5:2009(E): Biological evaluation of medical devices – Part 5: In vitro cytotoxicity studies (absorbance measurement in MTT assay). Cells were cultured at 1 x 10⁻⁶ cells according to the planned exposure time. 5 / ml, 5x10 4 / ml and 2.5x10 4 The culture was seeded at a density of 100 μl / ml into a 96-well plate.

[0235] Under standard conditions (5% CO2 and 37°C), cells were cultured overnight in supplemented DMEM medium to allow fibroblasts to diffuse to the bottom of the culture vessel. The assay employed a direct method, adding a purified protein solution reconstituted in the medium to the culture. After examining confluence and colony status, 100 μl of the protein solution from the medium was placed on plates at concentrations of 1, 0.5, and 0.1 mg / mL. Cell densities were set at 1 x 10⁻⁶ cells / mL. 5 Incubate at 24 hours with a plate temperature of 5 x 10⁶ ml. 4 / ml of culture was exposed to EJ17zipR protein for 48 hours, with an initial density of 2.5 x 10⁶. 4 / ml of cells were exposed to EJ17zipR solution for 72 hours.

[0236] Subsequently, the cells were incubated with MTT reagent solution for 2 hours, then all liquid above the cells was removed, and the formed formazan crystals were dissolved in DMSO. The amount of colored product formed was determined to be proportional to the number of viable cells by measuring absorbance at 570 and 650 nm wavelengths. According to standards, the expected result is that the cell viability exposed to the cytotoxic agent is not less than 70% of the viability of untreated cells in the negative control.

[0237] Based on the results, it was concluded that within the tested concentration range (0.1–1 mg / ml), EJ17zipR protein showed no cytotoxicity to the L-929 fibroblast cell line after exposure at 24, 48, and 72 hours. Figure 22 ).

[0238] Summary of bioactivity studies

[0239] Biological testing results indicate that the recombinant protein of this invention may become a valuable component of 3D printing bio-inks. Its applications are wide-ranging: as a coating agent for culture vessels in adherent cell cultures (replacing fibronectin or commercially available matrices); as a component of bio-inks for printing organs, organoids, and spheres; and as a factor promoting cell adhesion, proliferation, and migration in three-dimensional tissue models and complex vascular systems.

[0240] Example 7: The usefulness of biomaterials containing RE15mR protein in 3D bioprinting technology

[0241] Evaluation of the usefulness of materials containing methacrylamide gelatin (GelMa) and RE15mR protein.

[0242] RE15mR protein obtained from Escherichia coli BLR DE3 strain culture and RE15mR protein purified by lyophilization were used to manufacture a bio-ink with high practicality in 3D bioprinting technology for constructs. The bio-ink consists of two main components: (i) recombinant RE15mR protein with a molecular weight of 26 kDa, mainly containing arthropod elastin and elastin domains, synthesized as described above; and (ii) methacrylamide gelatin (GelMa DS 80; Polbionica Ltd., Poland), at a concentration of 5-20% (w / v) in 1xPBS, containing LAP as a photoinitiator, with a final concentration not exceeding 0.5% for cellular biomaterials, or 0.1% to 2% (optimally 0.5%) for non-cellular materials (Lithium phenyl-2,4,6-trimethylbenzoylphosphonite) (Polbionica Sp. z oo, Poland). The recombinant protein was used at four concentrations: 0.1, 0.5, 1.0, and 1.5 mg / ml. The reference sample was 10% GelMa containing LAP without the addition of recombinant protein.

[0243] The rheological properties of the developed material were tested using an Anton Paar MCR 72 rheometer. Three rheological parameters were measured. The complex modulus as a function of temperature was measured at 30% deformation, a frequency of 1 Hz, and a temperature range of 10–40°C. The complex modulus as a function of set strain from 0.01% to 100% was measured at a frequency of 1 Hz and a temperature of 20°C. Rotational measurements of dynamic viscosity were performed at a constant temperature of 20°C and a constant shear rate of 21 / s. All tests on the test material were conducted using a 25 mm diameter plate with the table containing the sample positioned 1 mm from the plate. The suitability of the characterized proteins for use as components in hydrogels or bioinks, which have high practical applications in 3D bioprinting, was investigated. Based on the rheological measurements, the dependence of the sol-gel phase transition point, storage modulus, and loss modulus on shear stress, as well as the average viscosity at a given temperature and constant shear rate, were determined. Figure 23 The rheological measurements of the test material based on GelMa containing the RE15mR recombinant protein are shown, where A - gel point, B - composite modulus, and C - dynamic viscosity.

[0244] The use of recombinant RE15mR protein in hydrogels did not affect the sol-gel phase transition temperature, which was 17°C for both the reference sample and the test material. However, the modulus values ​​changed due to variations in RE15mR protein concentration. Using high concentrations of recombinant RE15mR protein in hydrogels did not affect the dependence of the storage modulus on shear stress; the loss modulus was higher than the storage modulus, indicating that the viscous properties were superior to the elastic properties. Using relatively low protein concentrations resulted in an initial increase in the storage modulus relative to the loss modulus under low shear stress.

[0245] Adding recombinant RE15mR protein to the hydrogel leads to an increase in dynamic viscosity; the higher the protein concentration, the higher the viscosity of the biomaterial, which is important when using the material in bioprinting technology.

[0246] Compared to a reference bioink without added protein, the addition of recombinant RE15mR protein to a bioink containing dECM had no effect on dynamic viscosity and sol-gel phase transition point.

[0247] Compared to a reference bioink, using a relatively low concentration of 0.1 mg / ml RE15mR protein significantly increased the values ​​of the complex module components. An increase in modulus indicates enhanced elasticity or viscosity of a given material.

[0248] The printability of the developed biomaterials was tested. A specially developed three-stage evaluation system was used: a fusion test of fibers printed in template form, a collapse test of fibers printed on a 3D platform, and an evaluation of fiber continuity during printing with 3 ml of continuous bio-ink. Before testing, the prepared printing material was incubated at an appropriate temperature of 21–25°C (optimal time 15 min) in a heated block and a BIOX Cellink printhead. To select suitable material printing parameters, a series of extrusion tests were conducted under different temperature (10–30°C) and pressure (10–70 kPa) conditions.

[0249] Based on the obtained data, the optimal printing parameters for the given material were selected, including pressure in the range of 35-55 kPa, temperature in the range of 21-23°C, and printing speed in the range of 8-20 mm / s.

[0250] To conduct fiber fusion testing, a printed model was designed in which two layers of the test material were printed sequentially without the need for an external lamp to crosslink the layers. This was achieved using BIO X. TMThe printed parts were produced using an extrusion printer (Cellink, Sweden). The prepared prints followed a 0°–90° pattern to reproduce the 2D effect and increase the interfiber distance (FD). The interfiber distance ranged from 1–5 mm in 1 mm increments. The printing speed, needle diameter, and printing distance used in the tests were 20 mm / s, 21 G (0.609 mm), and 0.8 mm, respectively. The prints were crosslinked using an external UV-Vis lamp at 365 or 405 nm, with a power range of 13–28.5 W / cm². 2 The diffusion rate lasted for 15-20 seconds. Based on the results, two parameters were determined: the percentage diffusion rate (material spreading rate) (Dfr) and printability (Pr). The diffusion rate in pores without material spreading was 0 (i.e., At = Aa), while for a perfect model representation, the printability was 1.

[0251]

[0252] in:

[0253] D fr - Pore diffusion rate

[0254] P r - Printability of materials

[0255] A t - Theoretical pore surface

[0256] A a - Actual pore surface

[0257] L – Aperture circumference.

[0258] Figure 24 Results evaluating the printability of GelMa-based biomaterials containing the RE15mR recombinant protein are presented, with Part A detailing the results of fiber fusion tests as a measure of the material's printability and resolution. Based on the results, it is concluded that printability improves as the pattern aperture increases, the diffusion rate decreases. The material containing the RE15mR recombinant protein was characterized with high printing resolution, superior to the material without added protein. Due to its high printing resolution and the absence of uncontrolled diffusion, this material can be used as an additive to bioinks when printing relatively small, highly detailed objects is required. This material can be used to bioprint controlled drug delivery systems or tissue models with vascular systems for testing novel active substances or oncology drugs. The ability to print high-resolution models enables the printing of models with complex, branching vascular systems, enabling the bioprinting of organs, as well as 3D models used in regenerative medicine and transplantation, ensuring faster vascularization of the fabricated constructs and ensuring optimal gas exchange and nutrient supply, even for large 3D constructs.

[0259] The mid-span deflection of the suspended fibers was analyzed to determine the material's collapse tendency. For the experiment, a special platform consisting of seven supports with known spacings of 1, 2, 3, 4, 5, and 6 mm was designed and 3D printed. The five internal supports of the structure have dimensions of 2 × 10 × 6 mm. 3 The two edge supports measure 5 × 10 × 6 mm. 3 Using BIO X TM An extrusion printer (Cellink, Sweden) deposits single fibers of test material onto a platform using bioprinting technology and immediately photographs the printed parts. Prints are prepared at a speed of 20 mm / s using a 21 G (0.609 mm) nozzle. The collapse area factor (C) is... f The area is the percentage of the actual area to the theoretical area after the suspension fiber deflection, calculated using the following formula:

[0260]

[0261] in:

[0262] C f - Collapse area coefficient

[0263] A c a - Actual area under the curve

[0264] A c t - The theoretical area under the curve.

[0265] If the material is too viscous to retain the fibers between the two supports, the actual area is zero, and the collapse factor is 0. On the other hand, if the fibers do not collapse and form straight bridges between consecutive supports, then A... c t = A c a The coefficient is 100%.

[0266] Figure 24 Part B shows the results of the fiber collapse test, as a measure of its stability. Materials containing the RE15mR recombinant protein were characterized by fiber continuity and stability within the optimal printing parameter range. The collapse rate exceeded 80%. Using recombinant RE15mR protein as an additive to hydrogels or bio-inks can improve parameters representing the high resolution and stability of printable fibers.

[0267] Fiber continuity was evaluated using a 0 / 1 system when printing 2–3 ml of test bio-ink, with a value of 0 at fiber breakage and 1 at stretching. Materials containing the RE15mR recombinant protein were characterized by fiber continuity and stability within the optimal printing parameter range. Test materials were printable within the following parameter range: temperature: 10–30°C, pressure: 10–70 kPa, extrusion speed: 8–25 mm / s. However, the optimal printing parameters were within the following range: temperature: 21–23°C, pressure: 35–55 kPa, printing speed: 8–20 mm / s.

[0268] Finally, the test materials were characterized using mechanical parameters. The mechanical compressive strength of the samples was tested using static compression tests. For this purpose, an apparatus was designed and assembled, consisting of the following components: a computer with Axis FM software Pronterface installed, a force gauge and tripod drive, a tripod with electric drive and control, an Axis FB50 force gauge (maximum force 50 N) mounted on the tripod, and a print compression head. For the experiments, cylindrical samples with the following dimensions were designed: d - diameter 10 mm, h - height 5 mm (100% infill, cross-linked with an external UV-Vis lamp after each layer), and printed on a BIO X™ 3D extruder (Cellink, Sweden). All samples were initially loaded with a force ranging from 0 to 0.05 N. The samples were compressed at a constant rate of 10 mm / min at room temperature until the deformation reached 80%, with data points collected every 0.025 s. After the measurement, the data and graphs (force versus measurement time) were saved. Based on the results, the mechanical strength of the sample is calculated as the maximum stress (the ratio of force to the printed sample surface area), and the Young's modulus is calculated as the slope coefficient of a simple relationship between stress and deformation within the deformation range of 0.1-0.5. The commonly used yield strength R0.01 is the stress value that causes permanent deformation of the test sample (after unloading) equal to 0.01% of the measured height.

[0269] Measurement results as follows Figure 25As shown, the use of recombinant RE15mR protein in bio-inks leads to significant changes in the mechanical parameters of the printed constructs. Increased protein concentration in biomaterials results in increased structural mechanical strength. Compared to a reference sample, adding low concentrations of RE15mR protein to biomaterials results in a decrease in the Young's modulus of the printed constructs, while increasing the RE15mR protein concentration leads to a further increase in this parameter. Using RE15mR protein as a hydrogel component increases the mechanical parameters of the printed constructs, making it suitable for 3D model fabrication. Depending on the concentration of the added protein component, materials with different properties can be prepared. Due to its high affinity for endothelial cells, biomaterials containing RE15mR protein can be used to fabricate blood vessels, vascular prostheses or complete tissue models produced via bioprinting, and processes for bioprinting organs with vascular systems. Materials rich in RE15mR recombinant protein, with its properties, provide an ideal matrix for the cells constituting the vascular system and ensure high mechanical strength of the printed constructs, which is crucial in flow systems. Furthermore, the biomaterial is characterized by high printing resolution, and its lumen is characterized by a smooth inner surface, resulting in a significant reduction in coagulation levels upon contact with blood.

[0270] The water absorption capacity of the test material was tested. For this purpose, 200 μl of biomaterial was poured into a weighing dish, and water was absorbed using a wavelength of 365 or 405 nm and a power of 13-28.5 mW / c. m2 Crosslinking was performed using light, with each exposure lasting 20 seconds, repeated three times. The sample was weighed again to obtain the mass of the crosslinked material. 10 ml of deionized water was added to the dish and sealed with plastic wrap. The dish was left at room temperature for 24 hours. After 24 hours, the water was removed, the dish was dried, and the sample was weighed again. Then, 10 ml of water was added again, the dish was sealed with plastic wrap, and the sample was left at room temperature for another 24 hours. The weighing was repeated after 48 and 72 hours, and the deionized water was replaced. The water absorption rate (water content per mg of crosslinked material) can be calculated using the following formula:

[0271] in:

[0272] W N - Biomaterial mass at a given time point after soaking

[0273] W M – Quality of biomaterials after casting and gelation

[0274] Water absorption rate measurement results are as follows Figure 26 As shown, using a low concentration of RE15mR recombinant protein resulted in improved water absorption compared to the reference sample. However, a three-fold increase in protein concentration compared to the reference sample significantly reduced the water absorption of the biomaterial.

[0275] To assess the usefulness of materials containing dECM and RE15mR proteins.

[0276] RE15mR protein obtained from *Escherichia coli* BLR DE3 strain culture and lyophilized and purified RE15mR protein were used to prepare a bioink with high practicality in 3D bioprinting of constructs. The analyzed material is a bioink based on cell-free extracellular matrix (dECM) obtained from the pancreas, enriched with recombinant RE15mR protein. In addition to the dECM-based bioink (81.27 mg dECM / ml), the test material also contained methacrylamide gelatin (37.15 mg / ml), methacrylamide hyaluronic acid (5.57 mg / ml), and LAP (2.32 mg / ml). The recombinant RE15mR protein used in the experiment had a molecular weight of 26 kDa and mainly contained arthropod elastin and elastin domains, at concentrations of 0.1 mg / ml and 1.5 mg / ml, respectively. The recombinant RE15mR protein was dissolved in methacrylamide hyaluronic acid at an appropriate concentration. The reference sample was dECM-based material without added recombinant protein.

[0277] The rheological properties of the developed material were tested using an Anton Paar MCR 72 rheometer. The complex modulus as a function of temperature was measured at 5% deformation, a frequency of 1 Hz, and a temperature range of 10–35°C. The complex modulus as a function of 1–100% of a set strain was measured at a frequency of 1 Hz and a temperature of 20°C. Rotational measurements of dynamic viscosity were performed at a constant temperature of 25°C and a constant shear rate of 100 1 / s. All tests on the test material were conducted using a 25 mm diameter plate, with the table containing the sample positioned 1 mm from the plate. Based on these results, the dependence of the sol-gel phase transition point, storage modulus, and loss modulus on shear stress, as well as the average viscosity at a given temperature and constant shear rate, were determined. The rheological test results are as follows: Figure 27 As shown, A represents the gel point, B represents the complex modulus, and C represents the dynamic viscosity. The results indicate that, compared to reference bioink A, the addition of RE15mR recombinant protein had no effect on the dynamic viscosity and sol-gel phase transition point. However, the addition of 0.1 mg / ml of this protein significantly increased the values ​​of each component of the complex modulus compared to reference bioink A.

[0278] The printability of the developed biomaterials was tested. A specially developed three-stage evaluation system was used: a fusion test of fibers printed in template form, a collapse test of fibers printed on a three-dimensional platform, and an evaluation of fiber continuity during continuous printing with 3 ml of bio-ink. The detailed methods used to test the printability of the evaluated materials have been described above. Based on the obtained data, optimal printing conditions were selected for the given materials, including pressure in the range of 35–45 kPa, temperature in the range of 23–25°C, and printing speed in the range of 8–20 mm / s. Figure 28 Part A shows the results of the filament fusion test, serving as a measure of the material's printability and resolution. Based on the results, it was concluded that as the pattern aperture increases, the diffusion rate decreases and printability improves. The material containing the RE15mR recombinant protein was characterized by high printing resolution, performing better than the material without the additive.

[0279] Figure 28 Part B shows the results of the fiber collapse test as a measure of its stability. Materials containing the RE15mR recombinant protein were characterized by fiber continuity and stability. The collapse rate exceeded 80%. Within the optimal printing parameter range (temperature: 23-25°C and pressure: 35-45 kPa), the test materials were characterized by fiber continuity and smoothness.

[0280] Finally, the test material was characterized using its mechanical parameters. The mechanical compressive strength of the samples was tested using a static compression test. Similar to before, based on the results, the mechanical strength of the samples was calculated as the maximum stress (the ratio of force to the surface area of ​​the printed sample), and Young's modulus was calculated as the slope coefficient of the simple relationship between stress and deformation within a deformation range of 0.1–0.5. The commonly used yield strength R0.01 is the stress value that causes permanent deformation of the test sample (after unloading) equal to 0.01% of the measured height. The measurement results are as follows: Figure 29 As shown.

[0281] The use of recombinant RE15mR protein in bio-inks results in significant changes in the mechanical parameters of the printed constructs. Adding a relatively low concentration (0.1 mg / ml) of RE15mR protein to biomaterials leads to improved mechanical strength of the printed constructs compared to a reference sample. As before, it is recommended that RE15mR protein be used as an additive in bio-inks for bioprinting hard tissue models (e.g., bone, cartilage) and tissue models with vascular systems subjected to high stress and shear forces (e.g., vascular systems in 3D bioprinted models and organs; and vascular and other structural models subjected to various flow, pressure, or force (forcing the model to be flexible)).

[0282] The water absorption capacity of the test material was tested according to the aforementioned procedure. The water absorption rate measurement results are as follows: Figure 30 As shown in the figure. The results indicate that adding recombinant RE15mR protein improves water absorption compared to reference bioink A. For constructs such as bone scaffolds, the water absorption capacity is crucial because it reflects the efficiency of fluid absorption and nutrient transport to cells.

[0283] Example 8: The usefulness of biomaterials containing EJ17zipR protein in 3D bioprinting technology

[0284] Evaluation of the usefulness of materials containing methacrylamide gelatin (GelMa) and EJ17zipR protein.

[0285] Purified EJ17zipR protein obtained from Escherichia coli BLR DE3 strain culture, as well as lyophilized and purified EJ17zipR protein, were used to manufacture a bio-ink with high practicality in 3D bioprinting of constructs. The bio-ink consists of two main components: (i) 86 kDa recombinant EJ17zipR protein, primarily containing elastin and silk fibroin domains, synthesized independently as described above; and (ii) methacrylamide gelatin (GelMa DS 80; Polbionica sp. zo.o., Poland), at a concentration of 5-20% (w / v) in 1xPBS, containing LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphonite) as a photoinitiator, with a final concentration not exceeding 0.5% for mixed-cell biomaterials and 0.1% to 2% (optimal 0.5%) for cell-free materials (Polbionica sp. zo.o., Poland). Recombinant protein was used at four concentrations: 0.1, 0.5, and 1.0 mg / ml. The reference sample was 10% GelMa containing LAP without added recombinant protein.

[0286] The rheological properties were tested in a similar manner to those tested on GelMa-based hydrogels rich in RE15mR recombinant protein.

[0287] Based on the results, the dependence of the sol-gel phase transition point, storage modulus, and loss modulus on shear stress, as well as the average viscosity at a constant shear rate at a given temperature, were determined. Figure 31Rheological measurements of the test material based on GelMa containing recombinant protein EJ17zipR are shown, where A represents the gel point, B represents the composite modulus, and C represents the dynamic viscosity. The results indicate that the addition of the recombinant protein has no effect on the sol-gel phase transition temperature, which is 17°C for both the reference sample and the test material. The addition of recombinant protein EJ17zipR, regardless of concentration, alters the dependence of the composite modulus on shear stress. For the reference sample, the loss modulus is higher than the storage modulus across the entire strain range tested. However, for the biomaterial containing recombinant protein EJ17zipR, the initial storage modulus is higher than the loss modulus; increasing the set deformation causes this relationship to change, with viscosity (G'') beginning to exceed elasticity (G'). Compared to the reference sample, the addition of the recombinant protein reduces the dynamic viscosity.

[0288] The printability of the developed biomaterial was tested following the three-step process described above. Based on the obtained data, optimal printing conditions were selected for the given material, including a pressure range of 35-50 kPa, a temperature range of 20-22°C, and a printing speed range of 8-20 mm / s. Figure 32 Part A shows the results of the filament fusion test, serving as a measure of the material's printability and resolution. Based on the results, it is concluded that as the pattern aperture increases, the diffusion rate decreases and printability improves. Materials containing the EJ17zipR recombinant protein were characterized by high printing resolution, superior to materials without the additive; therefore, it is recommended that the EJ17zipR recombinant protein be used as an additive for biomaterials used in 3D bioprinting technology. Figure 32 Part B shows the results of the fiber collapse test, serving as a measure of its stability. Materials containing the EJ17zipR recombinant protein were characterized by fiber continuity and stability. The collapse rate exceeded 80%. Within the optimal printing parameter range (temperature: 20–22°C and pressure: 35–50 kPa), the tested materials exhibited fiber continuity and smoothness.

[0289] Finally, the test material was characterized by mechanical parameters determined using a static compression test, the procedure of which has been described above. Based on the test results, the mechanical strength of the sample was calculated as the maximum stress (the ratio of force to the surface area of ​​the printed sample), and the Young's modulus was calculated as the slope coefficient of a simple relationship between stress and deformation within the deformation range of 0.1-0.5. The commonly used yield strength R0.01 is the stress value that causes permanent deformation of the test sample (after unloading) equal to 0.01% of the measured height. The measurement results are as follows... Figure 33 As shown.

[0290] The use of recombinant EJ17zipR protein in hydrogels leads to significant changes in the mechanical parameters of the printed constructs. Compared to structures made from reference materials, increased protein concentration in biomaterials results in increased mechanical strength. Furthermore, materials containing 1 mg / ml of EJ17zipR protein do not break under applied force. Hydrogels containing EJ17zipR protein exhibit significant elasticity. Materials containing EJ17zipR protein are recommended additives for bioprinting in bioprinting of tissue models (e.g., cartilage, bone, and blood vessels) requiring significant mechanical strength and elasticity, which are exposed to varying intensities of pressure and shear forces over short periods. Moreover, this material has been successfully used to print tissue models of functional vascular systems exposed to relatively high stresses, particularly when studying cells within colonized vascular systems. The prepared protein is also a solution for tissue models / heart models and other soft organs requiring vascular systems (e.g., pancreas, liver, etc.).

[0291] The water absorption capacity of the test material was tested according to the aforementioned procedure. The water absorption rate measurement results are as follows: Figure 34 As shown in the figure. The results obtained indicate that using low concentrations of recombinant EJ17zipR protein does not affect water absorption, while using higher concentrations of recombinant EJ17zipR protein leads to an increase in water absorption compared to the reference biomaterial.

[0292] To assess the usefulness of materials containing dECM and EJ17zipR proteins.

[0293] Purified EJ17zipR protein obtained from Escherichia coli BLR DE3 strain culture, as well as lyophilized purified EJ17zipR protein, were used to prepare a bio-ink with high practicality in 3D bioprinting technology. The analyzed material was a bio-ink based on cell-free extracellular matrix (dECM) obtained from the pancreas and enriched with recombinant EJ17zipR protein. In addition to the dECM-based bio-ink (81.27 mg dECM / ml), the test material also contained methacrylamide gelatin (37.15 mg / ml), methacrylamide hyaluronic acid (5.57 mg / ml), and LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphonate) (2.32 mg / ml). The recombinant EJ17zipR protein, with a molecular weight of 86 kDa, mainly contains elastin and silk fibroin domains at concentrations of 0.1 and 1.0 mg / ml, respectively. The recombinant EJ17zipR protein was dissolved in methacrylamide hyaluronic acid at an appropriate concentration. The reference sample was material without the addition of recombinant protein.

[0294] The rheological properties of the developed dECM-based material were tested using the aforementioned procedure. Based on the test results, the dependence of the sol-gel phase transition point, storage modulus, and loss modulus on shear stress, as well as the average viscosity at a constant shear rate at a given temperature, were determined. Figure 35 The rheological properties of the dECM-based material enriched with EJ17zipR recombinant protein are shown, where A represents the gel point, B represents the complex modulus, and C represents the dynamic viscosity. The results indicate that, compared to reference bioink A, the addition of EJ17zipR recombinant protein has no effect on the dynamic viscosity and sol-gel phase transition point. However, compared to reference bioink A, the addition of this protein leads to an increase in the values ​​of each component of the complex modulus.

[0295] The printability of the developed biomaterials was tested following the previously described procedure. Based on the obtained data, optimal printing conditions were selected for the given materials, including pressure in the range of 35–40 kPa, temperature in the range of 23–25°C, and printing speed in the range of 8–20 mm / s. Figure 36 Part A shows the results of the filament fusion test, serving as a measure of the material's printability and resolution. Based on the results, it was concluded that as the pattern aperture increases, the diffusion rate decreases and printability improves. The material containing the EJ17zipR recombinant protein was characterized by high printing resolution, superior to the material without additives. Due to its high printing resolution and the absence of uncontrolled diffusion, this material can be used as an additive for bio-inks when printing relatively small objects with rich detail is required. This material can be used for bioprinting controlled drug delivery systems or tissue models with vascular systems. Figure 36 Part B shows the results of the fiber collapse test, serving as a measure of its stability. Materials containing recombinant proteins were characterized by fiber continuity and stability. The collapse rate exceeded 70%. Within the optimal printing parameter range (temperature: 23-25°C and pressure: 40 kPa), the test material exhibited continuity and smoothness of the printed fibers.

[0296] Finally, the mechanical parameters determined by static compression tests were used to evaluate the test material. Similar to before, based on the test results, the mechanical strength of the sample was calculated as the maximum stress (the ratio of force to the surface area of ​​the printed sample), and the Young's modulus was calculated as the slope coefficient of the simple relationship between stress and deformation within the deformation range of 0.1-0.5. The commonly used yield strength R0.01 is the stress value that causes permanent deformation of the test sample (after unloading) equal to 0.01% of the measured height. The mechanical parameter measurement results are as follows: Figure 37 As shown.

[0297] The use of recombinant EJ17zipR protein in bio-inks resulted in significant changes in the mechanical parameters of the printed constructs. Compared to the reference sample, the use of EJ17zipR protein at a concentration of 0.1 mg / ml improved the mechanical strength of the printed constructs. However, the use of recombinant EJ17zipR protein did not cause a significant change in the Young's modulus of the printed constructs compared to the reference sample.

[0298] The water absorption capacity of the test material was tested according to the previously described procedure. The water absorption rate measurement results are as follows: Figure 38 As shown, the addition of recombinant protein EJ17zipR improves water absorption compared to reference bioink A. For example, in bone scaffolds, the water absorption capacity of the construct is crucial because it reflects the efficiency of fluid absorption and nutrient transport to cells.

Claims

1. A recombinant RE15mR hybrid protein, comprising 3 arthropod elastin domains, 1 K+ domain, 3 arthropod elastin domains, 1 MMP domain, 3 elastin domains, 1 K+ domain, 4 elastin domains, 1 K+ domain, 3 elastin domains, 1 K+ domain, 4 elastin domains, 1 K+ domain, 3 elastin domains, 1 MMP domain, and 7 RGD domains.

2. The recombinant RE15mR hybrid protein according to claim 1, wherein the amino acid sequence contains 12-15% proline and / or 32-38% glycine.

3. The recombinant RE15mR hybrid protein according to claim 1 or 2, wherein the molecular weight of the protein is 26 kDa.

4. The recombinant RE15mR hybrid protein according to any one of claims 1-3, having the amino acid sequence of SEQ #3.

5. The recombinant RE15mR hybrid protein according to any one of claims 1-4, wherein the recombinant protein comprises a fragment thereof.

6. The recombinant RE15mR hybrid protein according to any one of claims 1-5, wherein it is at least partially methacrylated.

7. A recombinant DNA sequence selected from: A recombinant DNA sequence encoding the recombinant RE15mR heterozygous protein as defined in any one of claims 1-4. A recombinant DNA sequence comprising a region encoding a DNA sequence encoding the recombinant RE15mR heterozygous protein as defined in any one of claims 1-4, and A recombinant DNA sequence that hybridizes with a DNA sequence encoding the recombinant RE15mR hybrid protein as defined in any one of claims 1-4.

8. The recombinant DNA sequence according to claim 7, wherein the recombinant DNA sequence is at least 80% identical, more preferably at least 90% identical, and most preferably homologous to the recombinant DNA sequence of SEQ #1.

9. A carrier comprising A recombinant DNA sequence containing a region comprising the recombinant DNA sequence as defined in claim 7 or 8, and / or A recombinant sequence comprising a portion of a region containing the recombinant DNA sequence as defined in claim 7 or 8.

10. The vector according to claim 9, wherein the vector is a plasmid containing a promoter derived from phage T7, preferably selected from pET11a-d, pET15b, pET19b, pET28a-c(+), pET21a-d(+), pET22b(+), pET23a-d(+), pET25b(+), pET44a-c(+), pET46Ek / LIC, and most preferably pET11a.

11. A prokaryotic expression system transformed with a recombinant DNA sequence and / or a recombinant sequence comprising a region including the recombinant DNA sequence defined in claim 7 or 8, wherein the recombinant sequence comprises a portion of the region including the recombinant DNA sequence defined in claim 7 or 8.

12. The expression system according to claim 11, wherein the expression system is an Escherichia coli cell.

13. A recombinant EJ17zipR hybrid protein, comprising 1 ZIP domain, 56 elastin domains, 10 silk fibroin domains, 7 RGD domains, 56 elastin domains, 10 silk fibroin domains, and 7 RGD domains.

14. The recombinant EJ17zipR hybrid protein according to claim 13, wherein the amino acid sequence contains 12-17% proline and / or 20-30% glycine.

15. The recombinant EJ17zipR hybrid protein according to claim 13 or 14, wherein the molecular weight of the protein is 86 kDa.

16. The recombinant EJ17zipR hybrid protein according to any one of claims 13-15, having the amino acid sequence of SEQ #4.

17. The recombinant EJ17zipR heterozygous protein according to any one of claims 13-16, wherein the recombinant protein comprises a fragment thereof.

18. The recombinant EJ17zipR heterozygous protein according to any one of claims 13-17, wherein it is at least partially methacrylated.

19. A recombinant DNA sequence selected from: A recombinant DNA sequence encoding the recombinant EJ17zipR heterozygous protein as defined in any one of claims 13-16. A recombinant DNA sequence comprising a region containing a DNA sequence encoding the recombinant EJ17zipR heterozygous protein as defined in any one of claims 13-16, and A recombinant DNA sequence that hybridizes with a DNA sequence encoding the recombinant EJ17zipR hybrid protein as defined in any one of claims 13-16.

20. The recombinant DNA sequence according to claim 19, wherein the sequence is at least 80% identical, more preferably at least 90% identical, and most preferably homologous to the recombinant DNA sequence of SEQ #2.

21. A carrier comprising A recombinant DNA sequence containing a region comprising the recombinant DNA sequence defined in claim 19 or 20, and / or A recombinant sequence containing a portion of a region comprising the recombinant DNA sequence as defined in claim 19 or 20.

22. The vector according to claim 21, wherein the vector is a plasmid containing a promoter derived from phage T7, preferably selected from pET11a-d, pET15b, pET19b, pET28a-c(+), pET21a-d(+), pET22b(+), pET23a-d(+), pET25b(+), pET44a-c(+), pET46Ek / LIC, and most preferably pET11a.

23. A prokaryotic expression system transformed with a recombinant DNA sequence and / or a recombinant sequence comprising a region including the recombinant DNA sequence defined in claim 19 or 20, wherein the recombinant sequence comprises a portion of the region including the recombinant DNA sequence defined in claim 19 or 20.

24. The expression system according to claim 23, wherein the expression system is an Escherichia coli cell.

25. Use of a recombinant hybrid protein selected from the RE15mR protein as defined in any one of claims 1-6 and the EJ17zipR protein as defined in any one of claims 13-18 as a component of a bioprinting ink.

26. Use of a recombinant hybrid protein selected from the RE15mR protein as defined in any one of claims 1-6 and the EJ17zipR protein as defined in any one of claims 13-18 as a component of biomaterial.

27. Use of the recombinant DNA sequence as defined in claim 7 or 8, or the vector as defined in claim 9 or 10, or the prokaryotic expression system as defined in claim 11 or 12 for the preparation of the recombinant RE15mR hybrid protein as defined in any one of claims 1-6.

28. Use of the recombinant DNA sequence as defined in claim 19 or 20, or the vector as defined in claim 21 or 22, or the prokaryotic expression system as defined in claim 23 or 24 for the preparation of the recombinant EJ17zipR hybrid protein as defined in any one of claims 13-18.

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