FGF-2 polypeptide with improved stability, method for preparing FGF-2 polypeptide and application of FGF-2 polypeptide

Abstract

The present invention relates to fibroblast growth factor 2 (FGF-2) polypeptides which have improved stability as compared to wild-type FGF-2 while maintaining biological activity. The invention provides a preparation method and application thereof in biotechnology research and industrial application, medicine, pharmaceutical industry, cosmetics, clean meat industry, organoid generation and 3D cell culture models and other related applications.

Description

Technical Field

[0001] The present invention relates to fibroblast growth factor 2 (FGF-2) polypeptides having increased stability (especially thermal stability) compared to wild-type FGF-2 and their use in research and industrial applications, such as, biotechnology research, medicine, pharmaceutical industry, cosmetics, clean meat industry, organoid generation and 3D cell culture models, and other related applications. Background Art

[0002] Fibroblast growth factors (FGFs) are a family of cell-signaling proteins involved in a variety of processes, particularly as key factors in the normal development of animal cells. These growth factors bind to heparin and heparan sulfate and generally activate cell surface receptors.

[0003] Fibroblast growth factors that signal through FGF receptors (FGFRs) regulate fundamental developmental pathways, including the regulation of angiogenesis and wound repair. FGFRs are expressed on many different cell types and regulate key cellular processes, such as, proliferation, differentiation, and survival, which makes FGF signaling vulnerable to disruption by cancer cells.

[0004] FGFs are secreted glycoproteins that are generally readily sequestered into the extracellular matrix and cell surface by heparan sulfate proteoglycans (HPSGs). For cell signaling, FGFs are released from the extracellular matrix by heparanase, proteases, or specific FGF-binding proteins, and the released FGFs then bind to cell surface HPSGs. Cell surface HPSGs also stabilize FGF ligand-receptor interactions, forming a ternary complex with FGFRs.

[0005] FGF receptors signal as dimers, and ligand-dependent dimerization leads to conformational changes in the receptor structure, which activate the intracellular kinase domain, resulting in intermolecular transphosphorylation of the tyrosine kinase domain and the intracellular tail. Phosphorylated tyrosine residues on the receptor serve as docking sites for adaptor proteins, which can themselves be directly phosphorylated by FGFRs, leading to the activation of multiple signal transduction pathways.

[0006] The human FGF-2 gene encodes not one protein, but a complex group of isoforms. The secreted isoform is a single-chain non-glycosylated polypeptide of 154 amino acids. The amino acid sequence of human FGF-2 has 99% homology with the amino acid sequence of bovine FGF-2 and a high degree of homology with the amino acid sequences of sheep and rodent FGF-2, indicating strong sequence conservation of structure and function.

[0007] In biotechnological research, the stability of FGF-2 is widely regarded as a major issue in the development of useful pharmaceutical products or serum substitutes. Manufacturers typically state that reconstituted FGF-2 solutions are only stable for up to 12 months when stored at -20 °C or lower. The reconstituted FGF solution is only stable for approximately one week at 4 °C, and it is recommended to be used within 24 hours at ambient temperature (around 25 °C). Nevertheless, at 25 °C, the FGF-2 solution at a concentration of 72 μg / mL loses 50% of its function after only 4 minutes. As the storage temperature is increased to 37 °C, 42 °C, and 50 °C respectively, the functional half-lives are shortened to 37, 33, and 10 minutes.

[0008] One challenge is to maintain the biological activity of FGF-2. Most of these efforts are directed at maintaining the activity of FGF-2 in cell culture studies or developing sustained-release formulations of FGF-2 for tissue engineering applications. For example, methods for maintaining the stability and biological activity of FGF-2 include modulating ionic interactions in the solution and chemical modification of FGF-2.

[0009] Adding excipients to an aqueous solution of FGF-2 is one of the simplest methods to stabilize FGF-2 by modulating ionic interactions in the solution. Common strategies include complexing FGF-2 with its endogenous stabilizer, heparin or heparin-like polymers, or polycations. The ionic interaction between FGF-2 and the additive reduces the structural energy of the heparin-binding site, stabilizes the native conformation of FGF-2, and prolongs its biological activity in aqueous media. Unfortunately, this method has some drawbacks. For example, there are safety concerns because pharmaceutical-grade heparin is isolated from porcine intestine and bovine lung tissues, and heparin is vulnerable to batch-to-batch variations. This variability can induce immune responses or be contaminated or adulterated with natural and synthetic heparinoids, leading to anaphylactoid reactions and death. In addition, native heparin is susceptible to degradation by heparinase and desulfation, which may have an adverse effect on its ability to stabilize FGF-2.

[0010] Methods for prolonging the biological activity of FGF-2 in aqueous media include point mutations of the protein and covalent grafting of the protein to scaffold materials.

[0011] Mutant variants of the FGF-2 protein have been developed by aligning the wild-type FGF-2 protein sequence with a stable FGF-1 mutant sequence or by combining several individual stabilizing mutations identified by other researchers in the field.

[0012] Computer modeling helps to identify useful point mutations. Sequences important for protein function have been identified and contribute the most to the structural free energy. Based on sequence conservation analysis, mutations in regions that may impair protein function are avoided. Instead, mutations that may lead to a decrease in the free energy of the protein are promoted. The obtained FGF-2 mutants show the lowest energy and an increased functional half-life.

[0013] Despite some progress in the prior art, there is still a need to further improve the stability of FGF-2 products while maintaining their biological activity. The object of the present invention is to prepare FGF-2 variants with improved stability and to find an effective, scalable, and economically advantageous method for producing FGF-2 in high yield. Summary of the Invention

[0014] According to the prior art, the present invention addresses the drawbacks of these solutions. The present invention provides a thermostable polypeptide that has FGF-2 activity and has improved stability while maintaining biological activity compared to wild-type FGF-2.

[0015] In one aspect of the present invention, the product is a truncated thermostable FGF-2 polypeptide having an amino acid substitution characterized by the sequence SEQ ID NO: 3. According to the present invention, the FGF-2 polypeptide having SEQ ID NO: 3 has 80.6% sequence similarity with human FGF-2 (SEQ ID NO: 1) and 81.3% sequence similarity with bovine FGF-2 (SEQ ID NO: 2).

[0016] In another aspect of the present invention, the product is a thermostable FGF-2 polypeptide having an amino acid substitution characterized by the sequence SEQ ID NO: 4.

[0017] The object of the present invention also lies in other FGF-2 polypeptides derived from the amino acid sequence of SEQ ID NO: 3, for example, FGF-2 polypeptides having at least 90% sequence similarity, or at least 93% sequence similarity, or at least 95% sequence similarity with the sequence of the truncated thermostable FGF-2 polypeptide (SEQ ID NO: 3), FGF-2 polypeptides characterized by SEQ ID NOs: 5-12 or SEQ ID NO: 21 (including the substitution L63Y), or other FGF-2 polypeptides described below.

[0018] The present invention also aims at other FGF-2 polypeptides derived from the amino acid sequence of SEQ ID NO: 4, for example, FGF-2 polypeptides having at least 90% sequence similarity, or at least 93% sequence similarity, or at least 95% sequence similarity with the sequence of the thermostable FGF-2 polypeptide (SEQ ID NO: 4), for example, FGF-2 polypeptides characterized by SEQ ID NOs: 13 to 20.

[0019] The present invention also aims at dimers of FGF-2 polypeptides, comprising linkers, for example, GSS linkers or SUMO linkers.

[0020] The present invention also aims at dimers of FGF-2 polypeptides, characterized by SEQ ID NOs: 22-27. The dimer of the FGF-2 polypeptide may comprise two linking sequences characterized by SEQ ID NO: 3, or two linking sequences having at least 90%, or 93%, or 95% sequence similarity with SEQ ID NO: 3. The linker for linking the two sequences may be, for example, a GSS linker (e.g., 6xGSS or 10xGSS), as used in the dimeric FGF-2 polypeptide characterized by SEQ ID NOs: 22-23, or a SUMO linker characterized by SEQ ID NOs: 28-31, as used in the dimeric FGF-2 polypeptide characterized by SEQ ID NOs: 24-27.

[0021] The present invention also provides a method for preparing FGF-2 polypeptides and their uses in the pharmaceutical industry, cosmetics, clean meat industry, organoid generation and 3D cell culture models, and other related applications.

[0022] Experiments have shown that, compared with human FGF-2, the stability is improved while maintaining the biological activity. The biological activity was tested by cell growth experiments, comparing the biological effects of commercially available FGF-2 and FGF-2 products according to the present invention. According to the present invention, the thermal stability of FGF-2 polypeptides and the influence of long-term storage on the product stability were studied using nano differential scanning fluorimetry (nanoDSF). Brief Description of the Drawings

[0023] Figure 1 Shows the sequence alignment of wild-type FGF-2 homologs (human, bovine) and the FGF-2 products according to the present invention characterized by SEQ ID NO: 3 and SEQ ID NO: 4.

[0024] Figure 2SDS-PAGE analysis showing the expression of truncated thermostable FGF-2 characterized by SEQ ID NO: 3 in Escherichia coli BL21 strain. Expression was induced by adding 1 mM IPTG and carried out at 20 °C for 1, 3, 5, 8, 12, 18, and 24 hours.

[0025] Figure 3 SDS-PAGE analysis of truncated thermostable FGF-2 characterized by SEQ ID NO: 3 after purification on a HisTrap column. (1) Supernatant after sonication and centrifugation, (2) pellet after sonication and centrifugation, (3) flow-through after loading the supernatant onto the HisTrap column, (4) fraction after washing with buffer containing 10 mM imidazole, (5) fraction after washing with buffer containing 40 mM imidazole, (6) fraction after washing with buffer containing 150 mM imidazole.

[0026] Figure 4 Elution profile of truncated thermostable FGF-2 characterized by SEQ ID NO: 3 on a Superdex200 Increase 10 / 300. The line with the maximum peak at approximately 20.9 minutes represents conductivity. The upper line with the maximum peak at approximately 17.6 minutes is the absorbance at 280 nm, the middle line is the absorbance at 260 nm, and the bottom line is the absorbance at 450 nm.

[0027] Figure 5 Thermal stability of truncated thermostable FGF-2 characterized by SEQ ID NO: 3 analyzed by nanoDSF. Changes in tryptophan emission at 330 and 350 nm were monitored and the ratio of 330 / 350 nm was plotted against temperature. Representative thermal unfolding curve (top) and its first derivative analysis (bottom).

[0028] Figure 6 Stability of truncated thermostable FGF-2 characterized by SEQ ID NO: 3 after storage at 4 °C for 30 days.

[0029] Figure 7 Stability of truncated thermostable FGF-2 characterized by SEQ ID NO: 3 after freeze-thaw cycles.

[0030] Figure 8 Results of cell culture when comparing commercially available FGF-2 and truncated thermostable FGF-2 characterized by SEQ ID NO: 3.

[0031] Figure 9SDS-PAGE analysis showing the expression of thermostable FGF-2 polypeptides is presented. (1) Uninduced cells, (2) Control cells expressing truncated thermostable FGF2 - SEQ ID No: 3, (3) Cells expressing dimeric thermostable FGF-2 - SEQ ID No: 24, (4) Cells expressing dimeric thermostable FGF-2 - SEQ ID No: 25, (5) Cells expressing dimeric thermostable FGF-2 - SEQ ID No: 26, (6) Cells expressing dimeric thermostable FGF-2 - SEQ ID No: 27, (7) Cells expressing thermostable FGF-2 - SEQ ID No: 21, (8) Cells expressing thermostable FGF-2 - SEQ ID No: 22, (9) Cells expressing thermostable FGF-2 - SEQ ID No: 23.

[0032] Figure 10 A cell culture system according to the present invention is shown. Detailed Description

[0033] The present invention addresses the drawbacks of existing technology solutions by providing thermostable polypeptides having FGF-2 activity and improved stability compared to wild-type FGF-2. The present invention also provides methods for preparing FGF-2 polypeptides and their uses.

[0034] In one aspect of the present invention, the product is a thermostable FGF-2 polypeptide derived from bovine FGF-2 (SEQ ID No: 2) and comprises at least one amino acid substitution: R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, S121P.

[0035] In one aspect of the present invention, the product is a truncated thermostable FGF-2 polypeptide characterized by the sequence SEQ ID No: 3, Figure 1 . The sequence SEQ ID No: 3 is derived from the bovine FGF-2 polypeptide (SEQ ID No: 2) and has the following modifications: an N-terminal deletion of amino acids 1 - 20 and 9 amino acid substitutions, specifically R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, S121P. Compared to the human FGF-2 polypeptide (SEQ ID No: 1), the bovine FGF-2 (SEQ ID No: 2) and the truncated thermostable FGF-2 polypeptide (SEQ ID No: 3) have the modification S137P. The nucleotide sequence encoding the truncated thermostable FGF-2 was cloned into the pET30a(+) expression vector.

[0036] In another aspect of the present invention, the FGF-2 polypeptide can be derived from a bovine FGF-2 polypeptide (SEQ ID NO: 2) with amino acids 1-15 to 1-22 deleted at the N-terminus. These polypeptides can contain at least one amino acid substitution relative to SEQ ID NO: 2: R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E or S121P.

[0037] According to the present invention, the truncated thermostable FGF-2 polypeptide (SEQ ID NO: 3) has 80.6% sequence similarity with human FGF-2 (SEQ ID NO: 1) and 81.3% sequence similarity with bovine FGF-2 (SEQ ID NO: 2).

[0038] In another aspect of the present invention, the product is a thermostable FGF-2 polypeptide, characterized by the sequence SEQ ID NO: 4, Figure 1 . The nucleotide sequence encoding the thermostable FGF-2 was cloned into the pET30a(+) expression vector.

[0039] According to the present invention, the thermostable FGF-2 polypeptide (SEQ ID NO: 4) has 93.5% sequence similarity with human FGF-2 (sequence SEQ ID NO: 1) and 94.2% sequence similarity with bovine FGF-2 (sequence SEQ ID NO: 2).

[0040] The present invention also aims at other FGF-2 polypeptides derived from the amino acid sequence of SEQ ID NO: 3, for example, FGF-2 polypeptides having at least 90% sequence similarity, or at least 93% sequence similarity, or at least 95% sequence similarity with the sequence of the truncated thermostable FGF-2 polypeptide (SEQ ID NO: 3).

[0041] The present invention also aims at FGF-2 polypeptides derived from the amino acid sequence of SEQ ID NO: 3, which contain at least one amino acid substitution: R11L, V32T, E34D, H39F, L72Y, S74I, C76N, S89E or S101P.

[0042] The present invention also aims at FGF-2 polypeptides characterized by SEQ ID NO: 5-12 or SEQ ID NO: 21 (containing the substitution L63Y), or other FGF-2 polypeptides described below.

[0043] Another object of the present invention is other FGF-2 polypeptides derived from the amino acid sequence of SEQ ID NO: 4. For example, FGF-2 polypeptides having at least 90% sequence similarity, or at least 93% sequence similarity, or at least 95% sequence similarity to the sequence of the thermostable FGF-2 polypeptide (SEQ ID NO: 4), such as FGF-2 polypeptides characterized by SEQ ID NOs: 13 to 20. The thermostable FGF-2 polypeptide derived from the amino acid sequence of SEQ ID NO: 4 may comprise at least one amino acid substitution: R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E or S121P.

[0044] In another aspect of the present invention, the product is a dimer of an FGF-2 polypeptide comprising a linker (e.g., a GSS linker or a SUMO linker).

[0045] Another object of the present invention is a dimer of an FGF-2 polypeptide characterized by SEQ ID NOs: 22-27. The dimer of the FGF-2 polypeptide according to the present invention may comprise two linking sequences characterized by SEQ ID NO: 3 or two linking sequences having at least 90%, or 93%, or 95% sequence similarity to SEQ ID NO: 3. The linker for linking the two sequences may be, for example, a GSS linker (6xGSS or 10xGSS), as used in the dimeric FGF-2 polypeptide characterized by SEQ ID NOs: 22-23, or a SUMO linker characterized by SEQ ID NOs: 28-31, as used in the dimeric FGF-2 polypeptide characterized by SEQ ID NOs: 24-27.

[0046] According to the present invention, the thermostable FGF-2 polypeptide may comprise the amino acid substitutions R31L (arginine at position 31 is replaced by leucine) and H59F (histidine at position 59 is replaced by phenylalanine) in the amino acid sequence of the bovine FGF-2 polypeptide (SEQ ID NO: 2). In another aspect of the present invention, the thermostable FGF-2 polypeptide may comprise other amino acid substitutions in addition to the R31L and H59F substitutions in SEQ ID NO: 2 or SEQ ID NO: 4. In one aspect of the present invention, the thermostable FGF-2 polypeptide may comprise an R31 substitution with any suitable amino acid (e.g., isoleucine or valine) and an H59 substitution with any suitable amino acid (e.g., tryptophan or isoleucine).

[0047] Another object of the present invention is FGF-2 polypeptides (SEQ ID NOs: 5-12), derived from the amino acid sequence of SEQ ID NO: 3 and comprising the following two residues:

[0048] (i)Isoleucine, valine at position 11 of SEQ ID NO: 3 (i.e., R11I, R11V)

[0049] (ii)Tryptophan, isoleucine at position 39 of SEQ ID NO: 3 (i.e., H39W, H39I).

[0050] The object of the present invention also lies in the FGF-2 polypeptide (SEQ ID NOs: 13-20), which is derived from the amino acid sequence of SEQ ID NO: 4 and contains the following two residues:

[0051] (i)Isoleucine, valine at position 31 of SEQ ID NO: 4 (i.e., R31I, R31V)

[0052] (ii)Tryptophan, isoleucine at position 59 of SEQ ID NO: 4 (i.e., H59W, H59I).

[0053] The object of the present invention also lies in a thermostable FGF-2 polypeptide with a melting temperature (Tm) higher than or equal to 55 °C, or higher than or equal to 65 °C, or higher than or equal to 68 °C.

[0054] The melting temperature (Tm) of the thermostable FGF-2 polypeptide according to the present invention can be in the range of 65 to 80 °C, or in the range of 67 to 75 °C, or in the range of 68 to 72 °C.

[0055] The method according to the present invention may include the following steps:

[0056] - Transforming competent Escherichia coli cells with plasmid DNA carrying the relevant FGF-2 sequence;

[0057] - Selection and screening of the transformed cells;

[0058] - Culturing of the transformed cells;

[0059] - Induction of FGF-2 production;

[0060] - FGF2 detection;

[0061] - Isolation and purification of FGF-2;

[0062] - Characterization of FGF-2.

[0063] According to the method of the present invention, a bacterial expression host system (e.g., an Escherichia coli bacterial expression host system) can be used for producing FGF-2 polypeptide due to its low cost, well-known biochemistry and genetics, rapid growth, and good productivity. The bacterial expression system is ideal for the production of FGF-2 because FGF-2 is a relatively small and single-domain protein with a compact fold. In addition, the crystal structure of the FGF-FGFR-heparin complex and the molecular mechanism of FGF protein action indicate that the normal function of FGF protein does not require post-translational modification or cofactors. Previously, FGF protein purified from Escherichia coli showed biological activity. Therefore, the bacterial expression host system seems to be ideal for large-scale industrial production of FGF-2. The presence of rare codons can be addressed by codon optimization.

[0064] In one aspect of the present invention, the strain for producing the FGF-2 polypeptide according to the present invention can be an Escherichia coli strain, e.g., BL21, BL-21-Gold, or BL21-CodonPlus RIPL.

[0065] Competent cells for producing FGF-2 polypeptide are transformed with plasmid DNA carrying the relevant FGF-2 sequence, which encodes the FGF-2 polypeptide defined, for example, by sequence SEQ ID NO: 3 or SEQ ID NO: 4, or any other sequence according to the present invention, e.g., SEQ ID NOs: 5 to 27.

[0066] The transformed cells are spread on a solid selection medium, e.g., the cell suspension is spread on an LB (Luria-Bertani) agar plate supplemented with an antibiotic. Later, e.g., the next day, colonies are picked and transferred to a liquid selection medium, e.g., LB broth supplemented with an antibiotic. The cells are grown for several hours, e.g., overnight. Then the culture can be placed in fresh liquid selection medium, e.g., LB broth supplemented with an antibiotic, and incubated at a temperature in the range of 15°C to 45°C, or 20°C to 40°C, or 30°C to 37°C. As the antibiotic, for example, kanamycin or any other suitable antibiotic can be used.

[0067] When the optical density (OD 550)When it reaches within the range of 0.4 to 1.0, or within the range of 0.5 to 0.9, or within the range of 0.6 to 0.8, an inducer is added. For example, isopropyl β-D-thiogalactoside (IPTG), or any other suitable inducer can be added to the cell culture to induce the production of FGF-2. The concentration of IPTG can be within the range of 0.001 to 10 mM, or within the range of 0.01 to 1 mM, or within the range of 0.05 to 0.5 mM. The cell culture is incubated at a temperature within the range of 4°C to 40°C, or within the range of 15°C to 30°C, or within the range of 18°C to 25°C. After an appropriate incubation period, for example, within the range of 1 hour to 72 hours, or within the range of 2 hours to 48 hours, or within the range of 12 hours to 24 hours, the culture can be harvested, for example, by centrifugation at 13000g, for example, at room temperature.

[0068] The cell pellet can be resuspended in 150 μl of 1x lithium dodecyl sulfate (LDS) gel sample buffer and heated to a temperature that can be within the range of 60 to 100°C, or within the range of 80 to 98°C, or within the range of 90 to 95°C, and the duration can be within the range of 1 to 60 minutes, or within the range of 2 to 30 minutes, or within the range of 3 to 10 minutes.

[0069] The sample can be spun at 13000g, for example, at room temperature, and analyzed by, for example, gradient SDS-PAGE. The volume of the sample to be analyzed can be within the range of 1 μl to 15 μl, or within the range of 2 μl to 12 μl, or within the range of 5 μl to 10 μl.

[0070] The purification of the FGF-2 polypeptide according to the present invention can be carried out, for example, by the following procedure. Induced bacterial cells are harvested from 1 liter of culture, for example, by centrifugation at 5000g for 20 minutes. The cell pellet is lysed, for example, by adding lysozyme and by sonication. The lysate is clarified by centrifugation, for example, at 75000g for 30 minutes at 4°C and filtered through a 0.22 μm filter membrane. The clarified lysate is loaded onto a HisTrap column. The FGF-2 polypeptide is eluted with imidazole and purified by cation exchange chromatography, for example, using HiTrap SP Sepharose gel.

[0071] The FGF-2 polypeptide can be analyzed by gel filtration chromatography, using, for example, a Superdex200 Increase column (10 / 300). The thermal stability of the FGF-2 polypeptide can be analyzed, for example, by nanoDSF.

[0072] The long-term stability of the FGF-2 polypeptide according to the present invention can be carried out. For example, the FGF-2 polypeptide can be stored in a refrigerator, for example, stored at 4 °C for 30 days, and then the protein can be analyzed by size exclusion chromatography.

[0073] Stability analysis can be carried out after one or more freeze-thaw cycles of the FGF-2 polypeptide according to the present invention. For example, by the method described in Example 5.

[0074] The biological activity of the FGF-2 polypeptide according to the present invention can be tested, and the achieved cell growth can be determined. Different concentrations of FGF-2 can be tested. For example, the concentration of FGF-2 can be 100, 10 or 1 ng / ml.

[0075] Thermostable truncated FGF-2 (SEQ ID NO: 3) has a stronger biological effect than commercially available FGF-2. For example, when the concentrations of the prepared FGF-2 polypeptide are 100 ng / ml, 10 ng / ml and 1 ng / ml respectively, cell counts 1.5 times, 2.8 times and 1.7 times higher are observed.

[0076] The experimental conditions for testing biological activity can be as described in Example 6, or any other suitable conditions set according to the needs of those skilled in the art.

[0077] According to the present invention, the method for preparing truncated thermostable FGF-2 and other FGF-2 derivatives is suitable for large-scale production. The method has been verified, for example, on a 1-liter scale and provides approximately 10 - 40 mg of FGF-2 with very high purity, for example, 94%. The melting temperature (Tm) is determined and the effect of mutations (N-terminal deletion and 9 substitutions in the FGF-2 chain) on increasing the stability of FGF-2 is evaluated.

[0078] According to the present invention, the FGF-2 polypeptide has been prepared in high yield. The product shows excellent thermal stability and very good long-term stability at 4 °C. The product can be frozen and successfully recovered.

[0079] The FGF-2 polypeptide according to the present invention can be successfully used in biotechnological research and industrial applications, medicine, the pharmaceutical industry, cosmetics, the clean meat industry, organoid generation and 3D cell culture models, and other related applications. These products according to the present invention can be used, for example, to prepare cosmetics, for example, creams, gels, lotions for improving the visual appearance of the skin and for skin regeneration.

[0080] The FGF-2 polypeptide according to the present invention can be used in many biotechnological processes, for example, for cell culture or for the biotechnological production of many different types of desired compounds, for example, proteins, active pharmaceutical ingredients or antibodies.

[0081] The FGF-2 polypeptide according to the present invention can be used in the cell culture process to prepare cultured meat products for human consumption or as pet food. The FGF-2 polypeptide according to the present invention can be used as a component of the culture medium. As Figure 10 shown, these methods of cell culture can be carried out in the cell culture system 1. The cell culture system 1 can include at least one of the following: for example, a culture device 2 formed by a production bioreactor, a cell harvesting device 3, a central control unit 4, or a monitoring device 5.

[0082] The culture device 2 (e.g., a production bioreactor), the cell harvesting device 3, the central control unit 4, and / or the monitoring device 5 can be directly or indirectly connected and / or communicate with each other.

[0083] The cell harvesting device 3 can include a filtration device, a centrifugation device, or any other suitable device for harvesting cells.

[0084] Optionally, the system can further include, for example, a seed tank or a device for preparing a cultured meat composition ( Figure 10 not shown in the figure).

[0085] The thermostable FGF-2 polypeptide according to the present invention can be used as a component of the culture medium in many biotechnological processes, for example, in the culture of mammalian cells, for example, in order to prepare cultured meat products in the clean meat industry. The thermostable FGF-2 polypeptide according to the present invention can be used as a signaling compound in the culture medium. The culture medium according to the present invention can further contain amino acids or their sources, in combination with at least one type of compound, which can be selected from: sugars, fatty acids, vitamins and organic micronutrients, mineral compounds, supplements, for example, iron supplement compounds, organic amines, shear protectants, additional compounds, or any other suitable compounds.

[0086] Examples

[0087] Example 1: Preparation of a truncated thermostable FGF-2 polypeptide (SEQ ID No.: 3)

[0088] Competent cells of three selected E. coli strains (BL21, BL-21-Gold, and BL21-CodonPlus RIPL) were transformed with plasmid DNA of the thermostable truncated construct FGF-2 corresponding to SEQ ID No.: 3, and each cell suspension was plated on an agar plate supplemented with kanamycin. The next day, two colonies from each plate were picked into 5 mL of LB broth supplemented with kanamycin and grown overnight. The next day, 50 μL of each overnight culture was added to two fresh 5 mL of LB broth supplemented with kanamycin and incubated at 37 °C at a speed of 220 rpm. After 70 minutes, the cell culture was cooled to 25 °C, which took 20 minutes. IPTG with a final concentration of 1 mM was added to induce the production of thermostable truncated FGF-2. The uninduced cell culture was used as a control sample. The cell culture was incubated at 25 °C at a speed of 220 rpm for 24 hours. After the incubation period, 1 mL of each culture was pipetted into a clean microcentrifuge tube, and the cells were centrifuged at 13000 g at room temperature. The cell pellet was resuspended in 150 μL of 1x lithium dodecyl sulfate (LDS) gel sample buffer and heated to 95 °C for 5 minutes. The samples were centrifuged at 13000 g at room temperature, and 10 μL of each sample was loaded onto a gradient SDS-PAGE.

[0089] Figure 2 SDS-PAGE analysis of the expression of truncated thermostable FGF-2 characterized by SEQ ID No.: 3 in E. coli BL21 strain is described. Expression was induced by adding 1 mM IPTG and carried out at 20 °C for 1, 3, 5, 8, 12, 18, and 24 hours.

[0090] Truncated thermostable FGF-2 (SEQ ID No.: 3) was successfully expressed in all three E. coli strains BL21, BL21-Gold, and BL21-CodonPlus RIPL. In the IPTG-induced cell cultures, the FGF-2 protein was clearly visible between 15 and 20 kDa. No band corresponding to truncated thermostable FGF-2 was observed in the non-induced cells. The best yield was observed in BL21(DE3) cells.

[0091] Example 2: Preparation of truncated thermostable FGF-2 polypeptide (SEQ ID No.: 3) and its purification on HisTrap FF column and Superdex200 Increase column

[0092] BL21(DE3) competent cells were transformed with plasmid DNA of truncated construct FGF-2 and plated on agar plates supplemented with kanamycin. The next day, colonies were picked and grown overnight in 5 mL of LB broth supplemented with kanamycin. The next day, 1 mL of the overnight culture was added to 1 L of fresh LB broth supplemented with kanamycin. The cultures in 2 L conical flasks (2 x 0.5 L) were incubated at 37 °C at 220 rpm. When the cell density reached an OD550 of 0.6, the cultures were cooled to 25 °C on ice, which took approximately 5 minutes. IPTG at a final concentration of 1 mM was used to induce expression. After induction, the cells were grown at 25 °C at 220 rpm. Twenty-four hours after induction, the cells were harvested by centrifugation at 5000 g for 20 minutes. The cell pellet was resuspended in 100 mL of cold buffer containing 30 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)-Hepes (pH 7.5), 500 mM NaCl, 10 mM imidazole, 10 mM MgCl2, 1% NP-40, 1 tablet of protease inhibitor SigmaFast, DNAse (total 5 mg) and lysozyme (total 100 mg). The suspension was incubated on ice for 30 minutes. Then, the cells were lysed by sonication on ice for 8 minutes (10 seconds on / 20 seconds off, amplitude 40%) and the lysate was clarified by centrifugation at 75000 g for 30 minutes at 4 °C. The lysate was filtered through a 0.22 μm filter membrane and loaded onto a HisTrap FF (5 mL) column at a flow rate of 5 mL / min, which was equilibrated in 30 mM HEPES (pH 7.5), 500 mM NaCl and 10 mM imidazole. FGF-2 was washed with a buffer containing 30 mM HEPES (pH 7.5), 500 mM NaCl and 40 mM imidazole and finally eluted with 30 mM HEPES (pH 7.5), 500 mM NaCl and 150 mM imidazole. The protein was concentrated to 1 mL using a VivaSpin Turbo centrifugal concentrator with a molecular weight cut-off of 10 kDa and diluted 20-fold with a buffer containing 15 mM Hepes (pH 7.5). The diluted protein was loaded onto HiTrap SP Sepharose HP (5 mL) at a flow rate of 5 mL / min and eluted with a continuous salt gradient of a buffer containing 15 mM HEPES (pH 7.5) and 1 M NaCl. The total gradient elution time was 40 minutes and the gradient was set from 0% to 60% of 15 mM HEPES (pH 7.5) and 1 M NaCl. The flow rate was 3 mL / min. The protein was concentrated to 0.5 mL using a VivaSpin Turbo centrifugal concentrator with a molecular weight cut-off of 10 kDa and loaded onto a Superdex 200 Increase column (10 / 300).In a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl, isocratic elution was performed at a rate of 0.5 ml / min from a Superdex 200 Increase column.

[0093] SDS PAGE analysis was used to control the purification results of the truncated FGF-2 characterized by SEQ ID No.: 3 ( Figure 3 ). After sonication, the lysate was centrifuged, and 10 μl of the supernatant (1) and the pellet (2) were loaded onto the gel. Then the supernatant was loaded onto a HisTrap column, flowed through (3), fractions washed with a buffer containing 10 mM imidazole (4), 40 mM imidazole (5), and finally loaded onto the gel with 150 mM imidazole (6), and analyzed using SDS PAGE.

[0094] Figure 4 The elution curve of the truncated thermostable FGF-2 characterized by SEQ ID No.: 3 on a Superdex 200 Increase 10 / 300 is shown. The line with the maximum peak at approximately 20.9 minutes represents the conductivity. The upper line with the maximum peak at approximately 17.6 minutes is the absorbance at 280 nm, the middle line is the absorbance at 260 nm, and the bottom line is the absorbance at 450 nm.

[0095] Example 3: Thermostability analysis

[0096] The thermostability of FGF-2 was analyzed using nanoDSF in a Prometheus NT.48. In a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl, FGF-2 (1 mg / ml) was loaded into a standard NanoTemper capillary. Three measurements were performed at a heating rate of 1.5 °C / min from 20 °C to 90 °C. The change in tryptophan emission at 330 and 350 nm was monitored, and the ratio of 330 / 350 nm was plotted against temperature. In one aspect of the present invention, the Tm of the thermostable truncated FGF-2 corresponding to SEQ ID No.: 3 determined by nanoDSF was 68.4 °C ( Figure 5 ). A representative thermal unfolding curve (top) and its first derivative analysis, showing the melting temperature of the protein (bottom), are shown.

[0097] The Tm in the experiment was approximately 14.9 °C higher than that of human FGF-2 (SEQ ID No.: 1). This value indicates excellent thermostability, which is consistent with the contribution of individual substitutions.

[0098] Example 4: Long-term stability

[0099] The heat-stable truncated FGF-2 corresponding to SEQ ID No.: 3 was stored in a 4 °C refrigerator for 30 days, and the protein was analyzed by size exclusion chromatography in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl to detect protein aggregation or degradation. Isocratic elution was performed from a Superdex 200 Increase column at a flow rate of 0.5 ml / min in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl.

[0100] Figure 6 Depicted is the elution profile of the truncated FGF-2 characterized by SEQ ID No.: 3 on a Superdex 200 Increase 10 / 300 after storage at 4 °C for 30 days. The bottom line is the absorbance at 260 nm, and the upper line is the absorbance at 280 nm. The straight line above the absorbance at 280 nm is the system pressure, and the upper line represents the conductivity.

[0101] The heat-stable truncated FGF-2 corresponding to SEQ ID No.: 3 eluted as the main peak from the Superdex 200 Increase 10 / 300 column. The elution volume was 19 mL, which was comparable to the elution time observed on the first day of purification. No shift to higher molecular weight was observed. Two small peaks were detected at elution volumes of 20.1 mL and 21.1 mL, which may be degradation products. Peak integration analysis indicated that the two small peaks reflected 38% of the total FGF-2.

[0102] Long-term stability analysis showed that 38% of the FGF-2 decomposed after storage at 4 °C for 30 days.

[0103] Example 5: Stability analysis after freeze-thaw cycles

[0104] Stability analysis was performed on the heat-stable truncated FGF-2 characterized by SEQ ID No.: 3 after one freeze-thaw cycle.

[0105] FGF-2 (1 mg / ml) in a buffer containing 15 mM slightly Hepes (pH 7.5) and 150 mM NaCl was frozen in liquid nitrogen. After one hour, the protein was thawed and loaded onto a Superdex 200 Increase 10 / 300 column to detect protein aggregation or degradation. Isocratic elution was performed from a Superdex 200 Increase column at a flow rate of 0.5 mL / min in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl.

[0106] Figure 7Describes the elution profile of the heat-stable truncated FGF-2 corresponding to SEQ ID No: 3 on a Superdex200 Increase 10 / 300 column after freeze-thaw. The bottom line is the absorbance at 260 nm (lower main peak), and the upper line is (higher main peak). The straight line above the absorbance at 280 nm is the system pressure, and the upper line shows the conductivity.

[0107] The FGF-2 eluted from Superdex200 Increase 10 / 300 was obtained as the main peak. The elution volume was 19 mL, corresponding to the elution time observed on the first day of purification. No shift to higher molecular weights was observed. A small peak was observed at an elution volume of 21.1 mL, which may be a degradation product. Peak integration analysis indicated that the small peak reflected 10% of the total FGF-2.

[0108] After one freeze-thaw cycle, approximately 10% degradation of the heat-stable truncated FGF-2 was observed.

[0109] Example 6: Testing biological activity

[0110] The biological effects of the FGF-2 polypeptide according to the present invention (heat-stable truncated FGF-2, SEQ ID No: 3) have been tested and compared with commercially available FGF-2 ( Figure 8 ). Three different concentrations of FGF-2 were tested: 100, 10, and 1 ng / ml. In the case of commercially available FGF-2, the cell growth obtained generally decreased with decreasing FGF-2 concentration. In the case of the heat-stable truncated FGF-2 (SEQ ID No: 3), when concentrations of 100 and 10 ng / ml were applied, the cell growth obtained was comparable, while a concentration of 1 ng / ml resulted in a lower cell count.

[0111] The heat-stable truncated FGF-2 (SEQ ID No: 3) has a stronger biological effect than commercially available FGF-2. For example, when the concentrations of the prepared FGF-2 polypeptide were 100 ng / ml, 10 ng / ml, and 1 ng / ml respectively, cell counts 1.5-fold, 2.8-fold, and 1.7-fold higher were observed.

[0112] Experimental details of testing biological activity:

[0113] ● Cells: C2C12 (seeding density of 4,000 cells per well)

[0114] ● Culture vessels: 24-well plates

[0115] ● Basal medium: Essential 8 TM Basal medium

[0116] ● Tested FGF-2 variants:

[0117] ○ Without FGF-2

[0118] ○ Commercially available FGF-2 - concentrations of 1, 10, and 100 ng / ml; thermostable truncated FGF-2 (SEQ ID NO: 3) - concentrations of 1, 10, and 100 ng / ml

[0119] ● Culture time points:

[0120] ○ Day 0 - Seeding

[0121] ○ Day 3 - Cell counting

[0122] ○ Day 7 - Cell counting, end of experiment

[0123] Example 7: Preparation of other thermostable FGF-2 polypeptides according to the present invention (SEQ ID NOs: 5–27)

[0124] According to the procedure described in Example 1, the thermostable FGF-2 polypeptides according to the present invention corresponding to SEQ ID NOs: 5–27 have been prepared.

[0125] Figure 9 Shown is the SDS-PAGE analysis of the expression of thermostable FGF-2 polypeptides. (1) Uninduced cells, (2) Control cells expressing truncated thermostable FGF2 - SEQ ID NO: 3, (3) Cells expressing dimeric thermostable FGF-2 - SEQ ID NO: 24, (4) Cells expressing dimeric thermostable FGF-2 - SEQ ID NO: 25, (5) Cells expressing dimeric thermostable FGF-2 - SEQ ID NO: 26, (6) Cells expressing dimeric thermostable FGF-2 - SEQ ID NO: 27, (7) Cells expressing thermostable FGF-2 - SEQ ID NO: 21, (8) Cells expressing thermostable FGF-2 - SEQ ID NO: 22, (9) Cells expressing thermostable FGF-2 - SEQ ID NO: 23.

[0126] Industrial applicability

[0127] According to the present invention, the FGF-2 polypeptides with improved stability can be used in scientific research and many industrial applications, for example, biotechnological research, medicine, the pharmaceutical industry, cosmetics, the clean meat industry, organoid generation, and 3D cell culture models, as well as other related applications.

[0128] Sequence listing:

[0129] SEQ ID NO: 1

[0130] Human FGF-2

[0131] MAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS

[0132] SEQ ID No.: 2

[0133] Bovine FGF-2

[0134] MAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYSSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS

[0135] SEQ ID No.: 3

[0136] Truncated heat-stable FGF-2

[0137] FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS

[0138] SEQ ID No.: 4

[0139] Heat-stable FGF-2

[0140] MAAGSITTLPALPEDGGSGAFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS

[0141] SEQ ID No.: 5

[0142] FGF-2 polypeptide variant 5

[0143] FPPGHFKDPKILYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQY KLGPKTGPGQKAILFLPMSAKS

[0144] SEQ ID No.: 6

[0145] FGF-2 polypeptide variant 6

[0146] FPPGHFKDPKVLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQ YKLGPKTGPGQKAILFLPMSAKS

[0147] SEQ ID No.: 7

[0148] FGF-2 polypeptide variant 7

[0149] FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPWIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQ YKLGPKTGPGQKAILFLPMSAKS

[0150] SEQ ID No.: 8

[0151] FGF-2 polypeptide variant 8

[0152] FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPIIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQY KLGPKTGPGQKAILFLPMSAKS

[0153] SEQ ID No.: 9

[0154] FGF-2 polypeptide variant 9

[0155] FPPGHFKDPKILYCKNGGFFLRIHPDGRVDGTRDKSDPWIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQ YKLGPKTGPGQKAILFLPMSAKS

[0156] SEQ ID No.: 10

[0157] FGF-2 polypeptide variant 10

[0158] FPPGHFKDPKILYCKNGGFFLRIHPDGRVDGTRDKSDPIIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQY KLGPKTGPGQKAILFLPMSAKS

[0159] SEQ ID No.: 11

[0160] FGF-2 polypeptide variant 11

[0161] FPPGHFKDPKVLYCKNGGFFLRIHPDGRVDGTRDKSDPWIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQ YKLGPKTGPGQKAILFLPMSAKS

[0162] SEQ ID No.: 12

[0163] FGF-2 polypeptide variant 12

[0164] FPPGHFKDPKVLYCKNGGFFLRIHPDGRVDGTRDKSDPIIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQY KLGPKTGPGQKAILFLPMSAKS

[0165] SEQ ID No.: 13

[0166] FGF-2 polypeptide variant 13

[0167] MAAGSITTLPALPEDGGSGAFPPGHFKDPKILYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS

[0168] SEQ ID No.: 14

[0169] FGF-2 polypeptide variant 14

[0170] MAAGSITTLPALPEDGGSGAFPPGHFKDPKVLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS

[0171] SEQ ID No.: 15

[0172] FGF-2 polypeptide variant 15

[0173] MAAGSITTLPALPEDGGSGAFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPWIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS

[0174] SEQ ID No.: 16

[0175] FGF-2 polypeptide variant 16

[0176] MAAGSITTLPALPEDGGSGAFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPIIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS

[0177] SEQ ID No.: 17

[0178] FGF-2 polypeptide variant 17

[0179] MAAGSITTLPALPEDGGSGAFPPGHFKDPKILYCKNGGFFLRIHPDGRVDGTRDKSDPWIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS

[0180] SEQ ID No.: 18

[0181] FGF-2 polypeptide variant 18

[0182] MAAGSITTLPALPEDGGSGAFPPGHFKDPKILYCKNGGFFLRIHPDGRVDGTRDKSDPIIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS

[0183] SEQ ID No.: 19

[0184] FGF-2 polypeptide variant 19

[0185] MAAGSITTLPALPEDGGSGAFPPGHFKDPKVLYCKNGGFFLRIHPDGRVDGTRDKSDPWIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS

[0186] SEQ ID No.: 20

[0187] FGF-2 polypeptide variant 20

[0188] MAAGSITTLPALPEDGGSGAFPPGHFKDPKVLYCKNGGFFLRIHPDGRVDGTRDKSDPIIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS

[0189] SEQ ID No.: 21

[0190] FGF-2 polypeptide variant 21

[0191] FGF2_mut_trunc_L83Y

[0192] FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYYAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS

[0193] SEQ ID No.: 22

[0194] FGF-2 dimeric polypeptide variant 22

[0195] FGF2_mut_trunc_GSS6x

[0196] FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKSGSSGSSGSSGSSGSSGSSFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS

[0197] SEQ ID No.: 23

[0198] FGF-2 dimeric polypeptide variant 23

[0199] FGF2_mut_trunc_GSS10x

[0200] FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKSGSSGSSGSSGSSGSSGSSGSSGSSGSSGSSFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS

[0201] SEQ ID No.: 24

[0202] FGF-2 Dimeric Polypeptide Variant 24

[0203] FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKSSITVRVRDQTGEETFFKIKKTTKMQKVFETYATRKGVQVNSLRFLLDGDRITPDQTPKMLELEDQDQIDCVLFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS

[0204] SEQ ID No.: 25

[0205] FGF-2 Dimeric Polypeptide Variant 25

[0206] FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKSGGGGSSSITVRVRDQTGEETFFKIKKTTKMQKVFETYATRKGVQVNSLRFLLDGDRITPDQTPKMLELEDQDQIDCVLGGGGSSFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGP GQKAILFLPMSAKS

[0207] SEQ ID No.: 26

[0208] FGF-2 Dimeric Polypeptide Variant 26

[0209] FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKSPSPSPSSITVRVRDQTGEETFFKIKKTTKMQKVFETYATRKGVQVNSLRFLLDGDRITPDQTPKMLELEDQDQIDCVLPSPSPSFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQ KAILFLPMSAKS

[0210] SEQ ID No.: 27

[0211] FGF-2 Dimeric Polypeptide Variant 27

[0212] FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKSMDVPATTEDVKESAESITVRVRDQTGEETFFKIKKTTKMQKVFETYATRKGVQVNSLRFLLDGDRITPDQTPKMLELEDQDQIDCVLEQTGGKGHGQIGAAAQFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS

[0213] SEQ ID No.: 28

[0214] SUMO short

[0215] SITVRVRDQTGEETFFKIKKTTKMQKVFETYATRKGVQVNSLRFLLDGDRITPDQTPKMLELEDQDQIDCVL

[0216] SEQ ID No.: 29

[0217] SUMO short fl

[0218] GGGGSSSITVRVRDQTGEETFFKIKKTTKMQKVFETYATRKGVQVNSLRFLLDGDRITPDQTPKMLELEDQDQIDCVLGGGGSS

[0219] SEQ ID No.: 30

[0220] SUMO short rl

[0221] PSPSPSSITVRVRDQTGEETFFKIKKTTKMQKVFETYATRKGVQVNSLRFLLDGDRITPDQTPKMLELEDQDQIDCVLPSPSPS

[0222] SEQ ID No.: 31

[0223] SUMO long

[0224] MDVPATTEDVKESAESITVRVRDQTGEETFFKIKKTTKMQKVFETYATRKGVQVNSLRFLLDGDRITPDQTPKMLELEDQDQIDCVLEQTGGKGHGQIGAAAQ

Claims

1. A thermostable FGF-2 polypeptide, derived from bovine FGF-2 (SEQ ID NO: 2), comprising at least one amino acid substitution: R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E or S121P.

2. A thermostable FGF-2 polypeptide, derived from bovine FGF-2 polypeptide (SEQ ID NO: 2), characterized by an N-terminal deletion of amino acids 1-15 to 1-22.

3. The thermostable FGF-2 polypeptide according to claim 2, derived from bovine FGF-2 polypeptide (SEQ ID NO: 2), characterized by an N-terminal deletion of amino acids 1-20.

4. The thermostable FGF-2 polypeptide according to claim 3, comprising at least one amino acid substitution: R11L, V32T, E34D, H39F, L72Y, S74I, C76N, S89E or S101P.

5. A thermostable FGF-2 polypeptide having at least 90% sequence similarity to SEQ ID NO:

3.

6. The thermostable FGF-2 polypeptide according to claim 5, having at least 93% sequence similarity to SEQ ID NO:

3.

7. The thermostable FGF-2 polypeptide according to claim 5, comprising at least one amino acid of isoleucine or valine at position 11 of SEQ ID NO:

3.

8. The thermostable FGF-2 polypeptide according to claim 5, comprising at least one amino acid of tryptophan or isoleucine at position 39 of SEQ ID NO:

3.

9. The thermostable FGF-2 polypeptide according to claim 5, characterized by SEQ ID NO:

3.

10. A thermostable FGF-2 polypeptide having at least 90% sequence similarity to SEQ ID NO:

4.

11. The thermostable FGF-2 polypeptide according to claim 10, comprising at least one amino acid of isoleucine or valine at position 31 of SEQ ID NO:

4.

12. The thermostable FGF-2 polypeptide according to claim 10, comprising at least one amino acid of tryptophan or isoleucine at position 59 of SEQ ID NO:

4.

13. The thermostable FGF-2 polypeptide according to claim 10, comprising at least one amino acid substitution: R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E or S121P.

14. The thermostable FGF-2 polypeptide according to claim 10, characterized by SEQ ID NO:

4.

15. The thermostable FGF-2 polypeptide according to claim 1, characterized by the sequence SEQ ID NO: 5-21.

16. A dimeric thermostable FGF-2 polypeptide comprising a GSS linker or a SUMO linker.

17. A dimeric FGF-2 polypeptide according to claim 16, comprising at least one sequence from SEQ ID NO: 1 to SEQ ID NO: 21 and a linker.

18. The dimeric FGF-2 polypeptide according to claim 17, comprising SEQ ID NO:

3.

19. A dimeric FGF-2 polypeptide, characterized by the sequence SEQ ID NO: 22 to SEQ ID NO:

27.

20. A thermostable FGF-2 polypeptide with a melting temperature (Tm) of higher than or equal to 55 °C.

21. The thermostable FGF-2 polypeptide according to claim 20, with a melting temperature (Tm) of higher than or equal to 65 °C.

22. The thermostable FGF-2 polypeptide according to claim 20, with a melting temperature (Tm) of higher than or equal to 68 °C.

23. Use of the thermostable FGF-2 polypeptide according to any one of the preceding claims in the preparation of cultured meat products.

24. Use of the thermostable FGF-2 polypeptide according to claim 23 in the preparation of cultured meat products, wherein, The cell culture process is carried out in a cell culture system 1, which comprises at least one of the following: cell culture equipment 2, cell harvesting equipment 3, a central control unit 4, or monitoring equipment 5.

25. Use of the thermostable FGF-2 polypeptide according to claims 1 to 22 in cosmetics.

26. A culture medium comprising the thermostable FGF-2 polypeptide according to claims 1 to 22.

27. The culture medium according to claim 26, comprising a thermostable FGF-2 polypeptide characterized by SEQ ID NO: 3 to 27.

Images