FGF-2 polypeptides with improved stability, methods for preparing FGF-2 polypeptides and uses thereof

Abstract

The present invention relates to fibroblast growth factor 2 (FGF-2) polypeptides that have improved stability while maintaining biological activity compared to wild-type FGF-2. The present invention provides preparation processes and uses thereof in biotechnology research and industrial applications, pharmaceutical industry, cosmetics, clean meat industry, generation of organoids 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 that have improved stability, particularly thermostability, compared to wild-type FGF-2, and their uses in research and industrial applications such as biotechnology research, medicine, the pharmaceutical industry, cosmetics, the clean meat industry, the generation of organoids and 3D cell culture models, and other related applications. [Background technology]

[0002] Fibroblast growth factors (FGFs) are a family of cell signaling proteins that are involved in a variety of processes, particularly as critical components of normal animal cell development. These growth factors bind to heparin and heparan sulfate, usually activating cell surface receptors.

[0003] Fibroblast growth factors, which signal through FGF receptors (FGFRs), regulate fundamental developmental pathways, including angiogenesis and wound repair. Because FGFRs are expressed on many different cell types and regulate essential cellular processes such as proliferation, differentiation, and survival, FGF signaling is susceptible to subversion by cancer cells.

[0004] FGFs are secreted glycoproteins that are generally readily sequestered in the extracellular matrix as well as the cell surface by heparan sulfate proteoglycans (HPSGs). For intracellular signaling, FGFs are released from the extracellular matrix by heparinases, proteases, or specific FGF-binding proteins, and then bind to HPSGs on the cell surface. HPSGs on the cell surface also stabilize the interaction of FGF ligands with their receptors, forming a ternary complex with FGFRs.

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

[0006] The human FGF-2 gene does not encode a single protein, but rather a complex set of isoforms. The secreted isoform is a single, non-glycosylated polypeptide of 154 amino acids. The amino acid sequence of human FGF-2 is 99% identical to that of bovine FGF-2 and shows high homology with bovine and rodent FGF-2, suggesting strong sequence conservation in terms of structure and function.

[0007] It is widely recognized that the stability of FGF-2 is a major concern in the development of useful pharmaceuticals and serum replacement in biotechnology research. Manufacturers typically state that reconstituted FGF-2 solutions are stable for 12 months only when stored at -20°C or below. Reconstituted FGF solutions are recommended for use within 24 hours at ambient temperature (around 25°C), as they are only stable for approximately one week at 4°C. Nevertheless, a 50% loss of function was observed in a 72 μg / mL FGF-2 solution after only 4 minutes at 25°C. The functional half-life decreases to 37, 33, and 10 minutes when storage temperatures are increased to 37, 42, and 50°C, respectively.

[0008] One of the challenges is maintaining the biological activity of FGF-2. Most of these efforts have been directed toward sustaining FGF-2 activity for cell culture studies or developing sustained-release formulations of FGF-2 for tissue engineering applications. For example, approaches to maintaining the stability and biological activity of FGF-2 include modulating ionic interactions in solution and chemically modifying FGF-2.

[0009] The addition of excipients to a solution of FGF-2 is one of the simplest methods to stabilize FGF-2 by modulating ionic interactions in solution. Common strategies include complexing FGF-2 with its endogenous stabilizers, heparin or heparin-like polymers, or polycations. Ionic interactions between FGF-2 and the excipients reduce the structural energy of the heparin-binding site, stabilizing the native conformation of FGF-2 and prolonging its biological activity in aqueous media. Unfortunately, this method has several drawbacks. For example, pharmaceutical-grade heparin is isolated from porcine intestine or bovine lung tissue, and heparin is susceptible to batch-to-batch variability, raising safety concerns. This variability can lead to contamination or contamination with natural and synthetic heparinoids, which can elicit immune responses or lead to anaphylactoid reactions and death. Furthermore, natural heparin is susceptible to degradation and desulfurization by heparinase, which can adversely affect its effectiveness in stabilizing FGF-2.

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

[0011] FGF-2 protein variants have been developed by aligning the wild-type FGF-2 protein sequence with stabilized FGF-1 mutant sequences or by combining individual stabilized mutants identified by other workers in the field.

[0012] Identification of useful point mutations was performed with the aid of computer modeling. Sequences important for protein functionality and contributing most to the structural free energy were identified. After analyzing sequence conservation, mutations in regions likely to impair protein function were avoided. Conversely, mutations likely to decrease the protein's free energy were promoted. The resulting FGF-2 mutants exhibited the lowest energy and a longer functional half-life.

[0013] Despite technological advances, there is a need to further improve the stability of FGF-2 products while maintaining biological activity. The objective of the present invention is to prepare FGF-2 variants with improved stability and to find an efficient, scalable, and economically advantageous process for producing FGF-2 in high yields. Summary of the Invention [Problem to be solved by the invention]

[0014] The shortcomings of solutions in the art are overcome by the present invention, which provides thermostable polypeptides that have FGF-2 activity and have improved stability compared to wild-type FGF-2 while maintaining biological activity. [Means for solving the problem]

[0015] In one embodiment of the invention, the product is a truncated thermostable FGF-2 polypeptide having amino acid substitutions characterized by the sequence of SEQ ID NO: 3. According to the invention, the FGF-2 polypeptide having SEQ ID NO: 3 has 80.6% sequence identity to Homo sapiens FGF-2 (SEQ ID NO: 1) and 81.3% sequence identity to Bos taurus FGF-2 (SEQ ID NO: 2).

[0016] In another embodiment of the invention, the product is a thermostabilized FGF-2 polypeptide having amino acid substitutions characterized in the sequence of SEQ ID NO:4.

[0017] The subject of the present invention is also 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 identity, or at least 93% sequence identity, or at least 95% sequence identity to the sequence of a truncated thermostable FGF-2 polypeptide (SEQ ID NO: 3), FGF-2 polypeptides characterized by SEQ ID NOs: 5 to 12 or SEQ ID NO: 21 (containing the substitution L63Y), or other FGF-2 polypeptides as described below.

[0018] The subject of the present invention is also 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 identity, or at least 93% sequence identity, or at least 95% sequence identity to 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 subject of the present invention is also a dimeric variant of an FGF-2 polypeptide comprising a linker, such as a GSS linker or a SUMO linker.

[0020] The subject of the present invention is also a dimeric variant of an FGF-2 polypeptide characterized by SEQ ID NOs: 22 to 27. A dimeric variant of an FGF-2 polypeptide may comprise two linked sequences characterized by SEQ ID NO: 3, or two linked sequences having at least 90%, 93%, or 95% sequence identity to 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 dimeric FGF-2 polypeptides characterized by SEQ ID NOs: 22 to 23, or a SUMO linker characterized by SEQ ID NOs: 28 to 31 as used in dimeric FGF-2 polypeptides characterized by SEQ ID NOs: 24 to 27.

[0021] The present invention also provides methods for preparing FGF-2 polypeptides and their use in generating organoids and 3D cell culture models in the pharmaceutical, cosmetic, and clean meat industries, as well as other related applications.

[0022] Experiments have demonstrated improved stability while maintaining biological activity compared to Homo sapiens FGF-2. Biological activity was tested in cell proliferation experiments, and the biological effects of the FGF-2 product of the present invention were compared with commercially available FGF-2. According to the present invention, the thermal stability of the FGF-2 polypeptide was examined using nano-differential scanning fluorometry (nanoDSF), and the effect of long-term storage on the product stability was also investigated. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 shows a sequence alignment of wild-type FGF-2 homologues (Homo sapiens, Bos taurus) and FGF-2 products according to the invention characterized by SEQ ID NO: 3 and SEQ ID NO: 4. [Figure 2] Figure 1 shows an SDS-PAGE analysis of the expression in E. coli strain BL21 of a truncated thermostable FGF-2 characterized by SEQ ID NO: 3. Expression was induced by the addition of 1 mM IPTG and carried out at 20°C for 1, 3, 5, 8, 12, 18, and 24 hours. [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. [Figure 4] 1 shows the elution profile of truncated thermostable FGF-2 characterized by SEQ ID NO: 3 on a Superdex200 Increase 10 / 300 column. The line with the maximum peak at approximately 20.9 minutes represents conductivity. The upper line with a peak at approximately 17.6 minutes represents absorbance at 280 nm, the middle line represents absorbance at 260 nm, and the lower line represents absorbance at 450 nm. [Figure 5]Figure 1 shows the thermal stability of truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 analyzed by nanoDSF. Changes in tryptophan emission at 330 nm and 350 nm were monitored and the 330 / 350 nm ratio was plotted versus temperature. Representative thermal unfolding curve (top) and its first derivative analysis (bottom). [Figure 6] FIG. 1 shows the stability of truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 after storage at 4° C. for 30 days. [Figure 7] FIG. 1 shows the stability of truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 after freeze-thaw cycling. [Figure 8] FIG. 1 shows the results of cell culture comparing commercially available FGF-2 with a truncated thermostabilized FGF-2 characterized by SEQ ID NO:3. [Figure 9] 1 shows SDS-PAGE analysis of 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 dimeric thermostable FGF-2: SEQ ID NO: 22, (9) cells expressing dimeric thermostable FGF-2: SEQ ID NO: 23. [Figure 10] 1 shows a cell culture system according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description of the Invention The shortcomings of state-of-the-art solutions are overcome by the present invention, which provides thermostable polypeptides that have FGF-2 activity and increased stability compared to wild-type FGF-2 while maintaining biological activity. The present invention also provides methods for the preparation of FGF-2 polypeptides and uses thereof.

[0025] In one embodiment of the invention, the product is a thermostable FGF-2 polypeptide derived from Bos taurus FGF-2 (SEQ ID NO: 2) containing at least one of the following amino acid substitutions: R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, S121P.

[0026] In one embodiment of the present invention, the product is a truncated thermostable FGF-2 polypeptide characterized by the sequence of SEQ ID NO: 3, Figure 1. The sequence of SEQ ID NO: 3 is derived from the Bos taurus FGF-2 polypeptide (SEQ ID NO: 2) with the following modifications: deletion of amino acids 1-20 at the N-terminus and the addition of nine amino acid substitutions, specifically R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, and S121P. Compared to the Homo sapiens FGF-2 polypeptide (SEQ ID NO: 1), the Bos taurus FGF-2 (SEQ ID NO: 2) and the truncated thermostable FGF-2 polypeptide (SEQ ID NO: 3) have an S137P modification. The nucleotide sequence encoding the truncated thermostable FGF-2 was cloned into the pET30a(+) expression vector.

[0027] In other embodiments of the present invention, FGF-2 polypeptides may be derived from Bos taurus FGF-2 polypeptide (SEQ ID NO: 2) by deleting amino acids 1-15 through 1-22 at the N-terminus. These polypeptides may contain at least one of the following amino acid substitutions compared to SEQ ID NO: 2: R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, or S121P.

[0028] The truncated thermostable FGF-2 polypeptide (SEQ ID NO: 3) of the present invention has 80.6% sequence identity to Homo sapiens FGF-2 (SEQ ID NO: 1) and 81.3% sequence identity to Bos taurus FGF-2 (SEQ ID NO: 2).

[0029] In another embodiment of the present invention, the product is a thermostable FGF-2 polypeptide characterized by SEQ ID NO: 4, the sequence of Figure 1. The nucleotide sequence encoding thermostable FGF-2 was cloned into the pET30a(+) expression vector.

[0030] The thermostabilized FGF-2 polypeptide (SEQ ID NO: 4) of the present invention has 93.5% sequence identity to the Homo sapiens FGF-2 sequence (SEQ ID NO: 1) and 94.2% sequence identity to the Bos taurus FGF-2 (SEQ ID NO: 2).

[0031] Also of interest are 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 identity, or at least 93% sequence identity, or at least 95% sequence identity to the sequence of the truncated thermostable FGF-2 polypeptide (SEQ ID NO: 3). Also of interest are FGF-2 polypeptides derived from the amino acid sequence of SEQ ID NO: 3 that contain at least one of the following amino acid substitutions: R11L, V32T, E34D, H39F, L72Y, S74I, C76N, S89E, or S101P.

[0032] The object of the present invention is also an FGF-2 polypeptide characterized by SEQ ID NO: 5 to 12 or SEQ ID NO: 21 (containing the substitution L63Y), or any other FGF-2 polypeptide according to the following description.

[0033] The present invention also relates to other FGF-2 polypeptides derived from the amino acid sequence of SEQ ID NO: 4, such as FGF-2 polypeptides having at least 90% sequence identity, or at least 93% sequence identity, or at least 95% sequence identity to 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. Thermostable FGF-2 polypeptides derived from the amino acid sequence of SEQ ID NO: 4 may contain at least one of the amino acid substitutions R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, or S121P.

[0034] In yet another embodiment of the invention, the product is a dimeric variant of an FGF-2 polypeptide that includes a linker, such as a GSS linker or a SUMO linker.

[0035] The subject of the present invention is also a dimeric variant of an FGF-2 polypeptide characterized by SEQ ID NOs: 22 to 27. A dimeric variant of an FGF-2 polypeptide according to the present invention may comprise two linked sequences characterized by SEQ ID NO: 3, or two linked sequences having at least 90%, 93%, or 95% sequence identity with SEQ ID NO: 3. The linker for linking the two sequences may be, for example, a GSS linker (6xGSS or 10xGSS) as used in dimeric FGF-2 polypeptides characterized by SEQ ID NOs: 22 to 23, or a SUMO linker characterized by SEQ ID NOs: 28 to 31 as used in dimeric FGF-2 polypeptides characterized by SEQ ID NOs: 24 to 27.

[0036] Thermostable FGF-2 polypeptides according to the present invention may comprise the amino acid substitutions R31L (arginine at position 31 substituted with leucine) and H59F (histidine at position 59 substituted with phenylalanine) in the amino acid sequence of Bos taurus FGF-2 polypeptide (SEQ ID NO: 2). In yet another aspect of the invention, thermostable FGF-2 polypeptides may comprise other amino acid substitutions other than the R31L and H59F substitutions in SEQ ID NO: 2 or SEQ ID NO: 4. In one aspect of the invention, thermostable FGF-2 polypeptides may comprise an R31 substitution with any suitable amino acid, for example, isoleucine or valine, and an H59 substitution with any suitable amino acid, for example, tryptophan or isoleucine.

[0037] The subject of the present invention is also an FGF-2 polypeptide (SEQ ID NOs: 5 to 12) derived from the amino acid sequence of SEQ ID NO: 3 and consisting of the following two residues: (i) Isoleucine, valine at position 11 of SEQ ID NO: 3 (i.e., R11I, R11V) (ii) Tryptophan, isoleucine at position 39 of SEQ ID NO: 3 (i.e., H39W, H39I).

[0038] The subject of the present invention is also an FGF-2 polypeptide (SEQ ID NOs: 13 to 20) derived from the amino acid sequence of SEQ ID NO: 4 and consisting of the following two residues: (i) Isoleucine, valine at position 31 of SEQ ID NO: 4 (i.e., R31I, R31V) (ii) Tryptophan, isoleucine at position 59 of SEQ ID NO: 4 (i.e., H59W, H59I).

[0039] Also an object of the present invention is a thermostabilized FGF-2 polypeptide having a melting temperature (Tm) of 55°C or higher, or 65°C or higher, or 68°C or higher.

[0040] The melting temperature (Tm) of the thermostabilized FGF-2 polypeptide according to the present invention may 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.

[0041] The method according to the invention comprises the following steps: -Transformation of competent E. coli cells with plasmid DNA carrying the relevant FGF-2 sequence -Selection and screening of transformed cells -Culture of transformed cells -Induction of FGF-2 production -FGF-2 detection -Isolation and purification of FGF-2 -FGF-2 characterization.

[0042] According to the methods of the present invention, bacterial expression host systems, such as Escherichia coli (E. coli) expression host systems, can be used to produce FGF-2 polypeptides due to their low cost, well-known biochemistry and genetics, rapid growth, and good productivity. Because FGF-2 is a relatively small, single-domain protein with a compact fold, bacterial expression systems are ideal for FGF-2 production. Furthermore, the crystal structure of the FGF-FGFR-heparin complex and the molecular mechanism of action of FGF proteins suggest that post-translational modifications and cofactors are not required for their proper function. Previously, FGF proteins purified from E. coli demonstrated biological activity. Therefore, bacterial expression host systems appear to be ideal for large-scale industrial production of FGF-2. The presence of rare codons can be addressed by codon optimization.

[0043] In one embodiment of the present invention, the strain used for the production of FGF-2 polypeptides according to the present invention may be an E. coli strain such as, for example, BL21, BL-21-Gold or BL21-CodonPlus RIPL.

[0044] Competent cells for FGF-2 polypeptide production are transformed with plasmid DNA containing a related FGF-2 sequence, for example encoding an FGF-2 polypeptide as defined by the sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or other sequences according to the invention, for example SEQ ID NOs: 5-27.

[0045] The transformed cells are plated on a solid selective medium. For example, a cell suspension is plated on a Luria-Bertani (LB) agar plate supplemented with an antibiotic. Colonies are picked later, e.g., the next day, and transferred to a liquid selective medium, such as LB broth supplemented with an antibiotic. The cells are cultured for several hours, e.g., overnight. The culture is then transferred to fresh liquid selective medium, e.g., LB broth supplemented with an antibiotic, and cultured at a temperature ranging from 15 to 45°C, or from 20 to 40°C, or from 30 to 37°C. The antibiotic may be, for example, kanamycin or another suitable antibiotic.

[0046] Optical density (OD) at 550 nm in the range of 0.4 to 1.0, or in the range of 0.5 to 0.9, or in the range of 0.6 to 0.8 550 When the cell culture reaches the FGF-2 concentration (K), an inducer, such as isopropyl β-D-thiogalactoside (IPTG) or any other suitable inducer, can be added to the cell culture to induce FGF-2 production. The IPTG concentration can be in the range of 0.001 to 10 mM, or 0.01 to 1 mM, or 0.05 to 0.5 mM. The cell culture is incubated at a temperature in the range of 4 to 40°C, or 15 to 30°C, or 18 to 25°C. After an appropriate culture period, for example, in the range of 1 to 72 hours, or 2 to 48 hours, or 12 to 24 hours, the culture can be harvested by centrifugation, for example, at 13,000 g, for example, at room temperature.

[0047] The cell pellet may be resuspended in 150 μl of 1× lithium dodecyl sulfate (LDS) gel sample buffer and heated to a temperature that may range from 60 to 100°C, or from 80 to 98°C, or from 90 to 95°C, for a time that may range from 1 to 60 minutes, or from 2 to 30 minutes, or from 3 to 10 minutes.

[0048] The samples can be centrifuged, for example, at 13000 g at room temperature and analyzed, for example, by gradient SDS-PAGE. The volume of the sample analyzed can range from 1 μl to 15 μl, or from 2 μl to 12 μl, or from 5 μl to 10 μl.

[0049] Purification of the FGF-2 polypeptide according to the present invention can be carried out, for example, by the following procedure: Bacterial cells after induction from a one-liter culture are harvested, for example, by centrifugation at 5,000 g for 20 minutes. The cell pellet is lysed, for example, by the addition of lysozyme and sonication. The lysate is clarified, for example, by centrifugation at 75,000 g for 30 minutes at 4° C. and filtered through a 0.22 μm 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.

[0050] The FGF-2 polypeptide can be analyzed by, for example, gel filtration chromatography using a Superdex200 Increase column (10 / 300). The thermal stability of the FGF-2 polypeptide can be analyzed by, for example, nanoDSF.

[0051] The long-term stability of the FGF-2 polypeptides according to the present invention may be evaluated. For example, the FGF-2 polypeptides may be stored in a refrigerator, e.g., at 4°C, for 30 days, and the protein may then be analyzed by size exclusion chromatography.

[0052] Stability analysis of FGF-2 polypeptides according to the invention after one or more freeze-thaw cycles may be performed, for example, by the method described in Example 5.

[0053] The biological activity of the FGF-2 polypeptides according to the invention may be tested and the cell proliferation achieved may be measured. Different concentrations of FGF-2 may be tested, for example, the concentration of FGF-2 may be 100, 10 or 1 ng / ml.

[0054] The thermostabilized truncated FGF-2 (SEQ ID NO: 3) exhibited stronger biological effects 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, 1.5-fold, 2.8-fold, and 1.7-fold higher cell numbers were observed, respectively.

[0055] The experimental conditions for testing biological activity may be as described in Example 6, or any other suitable conditions established according to the needs of those skilled in the art.

[0056] The method for preparing truncated thermostable FGF-2 and other FGF-2 derivatives according to the present invention is suitable for large-scale production. This method has been tested, for example, on a 1 liter scale, yielding approximately 10-40 mg of FGF-2 with very high purity, e.g., 94%. Melting temperatures (Tm) were measured, and the effects of mutations (deletions at the N-terminus and nine substitutions in the FGF-2 chain) that increase FGF-2 stability were assessed.

[0057] According to the present invention, FGF-2 polypeptide is prepared in high yield. The product exhibits excellent thermal stability and very good long-term stability at 4° C. The product can be frozen and successfully recovered.

[0058] The FGF-2 polypeptides according to the present invention can be successfully used in biotechnology research and industrial applications, medicine, pharmaceutical industry, cosmetics, clean meat industry, organoid and 3D cell culture model production, and other related applications.These products according to the present invention can be used, for example, to prepare cosmetic products such as creams, gels, and lotions for improving the visual appearance of the skin and rejuvenating the skin.

[0059] The FGF-2 polypeptides according to the invention can be used in many biotechnological processes, for example for cell culture or for the biotechnological production of many different kinds of required compounds, such as proteins, drug substances or antibodies.

[0060] The FGF-2 polypeptides according to the present invention can be used in cell culture processes for the purpose of preparing cultured meat products for human consumption or as pet food. The FGF-2 polypeptides according to the present invention can be used as a component of the culture medium. These cell culture processes can be carried out in a cell culture system 1, as shown in FIG. 10. The cell culture system 1 can include at least one of a culture device 2 formed by, for example, a production bioreactor, a cell harvesting device 3, a central control unit 4, or a monitoring device 5.

[0061] The culture device 2, eg, production bioreactor, cell harvesting device 3, central control unit 4, and / or monitoring device 5 may be directly or indirectly connected and / or in communication with each other.

[0062] The cell harvester 3 may include a filtering device, a centrifuge device, or any other suitable device for harvesting cells.

[0063] Optionally, the system may further include, for example, a seed tank or an apparatus for preparing the cultured meat composition (not shown in FIG. 10).

[0064] The thermostable FGF-2 polypeptides according to the present invention can be used as a component of culture media in biotechnology processes, such as mammalian cell culture, for example, for the purpose of preparing cultured meat products in the clean meat industry. The thermostable FGF-2 polypeptides according to the present invention can be used as a signaling compound in the culture media. The culture media according to the present invention can further comprise an amino acid or a source thereof in combination with at least one compound which can be selected from the group including sugars, fatty acids, vitamins and organic micronutrients, mineral compounds, supplements such as iron-replete compounds, organic amines, shear protectants, additional compounds, or any other suitable compound. [Example]

[0065] Example 1: Preparation of a truncated thermostable FGF-2 polypeptide (SEQ ID NO: 3) Competent cells of three selected Escherichia coli (E. coli) strains (BL21, BL-21-Gold, and BL21-CodonPlus RIPL) were transformed with plasmid DNA of the thermostable truncated FGF-2 construct corresponding to SEQ ID NO:3, and each cell suspension was plated onto an agar plate supplemented with kanamycin. Two colonies from each plate were transferred to 5 mL of LB medium supplemented with kanamycin and grown overnight. The next day, 50 μL of each overnight culture was added to two 5 mL tubes of fresh LB broth supplemented with kanamycin and incubated at 37°C and 220 rpm. After 70 minutes, the cell cultures were cooled to 25°C, which took 20 minutes. IPTG was added to a final concentration of 1 mM to induce production of the thermostable truncated FGF-2. An uninduced cell culture served as a control. The cell cultures were incubated at 220 rpm at 25°C for 24 hours. After the incubation period, 1 mL of each culture was pipetted into a clean microcentrifuge tube and centrifuged at 13,000 g at room temperature. The cell pellet was resuspended in 150 μL of 1× lithium dodecyl sulfate (LDS) gel sample buffer and heated to 95°C for 5 minutes. The samples were centrifuged at 13,000 g at room temperature, and 10 μl from each sample was loaded onto a gradient SDS-PAGE gel.

[0066] 2 shows an SDS-PAGE analysis of the expression of truncated thermostable FGF-2 characterized by SEQ ID NO: 3 in E. coli strain BL21. Expression was induced by the addition of 1 mM IPTG and carried out at 20° C. for 1, 3, 5, 8, 12, 18, and 24 hours.

[0067] Truncated thermostable FGF-2 (SEQ ID NO: 3) was successfully expressed in all three E. coli strains, BL21, BL21-Gold, and BL21-CodonPlusRIPL. FGF-2 protein was clearly visible between 15 kDa and 20 kDa in cell cultures induced with IPTG. No band corresponding to truncated thermostable FGF-2 was observed in uninduced cells. The highest yield was observed in BL21(DE3) cells.

[0068] Example 2: Preparation and purification of a truncated thermostable FGF-2 polypeptide (SEQ ID NO: 3) on a HisTrap FF column and a Superdex200 Increase column BL21(DE3) competent cells were transformed with the plasmid DNA of the 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 following day, 1 mL of the overnight culture was added to 1 L of fresh LB broth supplemented with kanamycin. The cultures (2 x 0.5 L) in 2 L Erlenmeyer flasks were incubated at 37°C and 220 rpm. When the cell density reached an OD550 of 0.6, the culture was cooled to 25°C on ice. This process took approximately 5 minutes. Expression was induced with a final concentration of 1 mM IPTG. After induction, the cells were grown at 25°C and 220 rpm. 24 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, one tablet of the protease inhibitor SigmaFast, DNAse (5 mg total), and lysozyme (100 mg total). The suspension was incubated on ice for 30 minutes. The cells were then lysed by sonication (10 sec on / 20 sec off, 40% amplitude) on ice for 8 minutes, and the lysate was clarified by centrifugation at 75,000 g for 30 minutes at 4°C. The lysate was filtered through a 0.22 μm membrane and loaded onto a 5 mL HisTrap FF column equilibrated in 30 mM HEPES (pH 7.5), 500 mM NaCl, and 10 mM imidazole at a flow rate of 5 mL / min. 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 10 kDa cutoff and diluted 20-fold with a buffer containing 15 mM Hepes (pH 7.5).The diluted protein was loaded onto a HiTrap SP Sepharose HP (5 mL) at a flow rate of 5 mL / min and eluted with a continuous salt gradient of buffer containing 15 mM HEPES (pH 7.5) and 1 M NaCl. The total gradient elution time was 40 min, with the gradient 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 10 kDa cutoff and loaded onto a Superdex200 Increase column (10 / 300). Isocratic elution from the Superdex200 Increase column was performed at 0.5 mL / min in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl.

[0069] The results of the purification of truncated FGF-2 characterized by SEQ ID NO: 3 were controlled using SDS-PAGE analysis (Figure 3). After sonication, the lysate was centrifuged, and 10 μl of the supernatant (1) and pellet (2) were loaded onto a gel. The supernatant was then loaded onto a HisTrap column, and the flow-through (3) and fractions after washing with a buffer containing 10 mM imidazole (4), 40 mM imidazole (5), and finally 150 mM imidazole (6) were loaded onto a gel and analyzed using SDS-PAGE.

[0070] The elution profile of truncated thermostabilized FGF-2 featuring SEQ ID NO:3 on Superdex200 Increase 10 / 300 is shown in Figure 4. The line with a maximum peak at approximately 20.9 minutes represents conductivity. The top line with a peak at approximately 17.6 minutes represents absorbance at 280 nm, the middle line represents absorbance at 260 nm, and the bottom line represents absorbance at 450 nm.

[0071] Example 3: Thermostability assay A thermal stability assay of FGF-2 was performed using nanoDSF in a Prometheus NT.48 (NanoTemper). Standard NanoTemper capillaries were loaded with FGF-2 (1 mg / ml) in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl. Measurements were performed in triplicate at temperatures from 20°C to 90°C with a temperature ramp of 1.5°C / min. Changes in tryptophan emission at 330 nm and 350 nm were monitored, and the 330 / 350 nm ratio was plotted versus temperature. In one embodiment of the present invention, the Tm of the thermostabilized truncated FGF-2 corresponding to SEQ ID NO:3, as measured by nanoDSF, was 68.4°C (Figure 5). A representative thermal unfolding curve (top) and its first derivative analysis (bottom) showing the melting temperature of the protein are shown.

[0072] The experimental Tm was approximately 14.9°C higher than that of Homo sapiens FGF-2 (SEQ ID NO: 1), indicating superior thermal stability and consistent with the contribution of individual substitutions.

[0073] Example 4: Long-term stability The thermostabilized truncated FGF-2 corresponding to SEQ ID NO:3 was stored in a refrigerator at 4°C for 30 days after production, and the protein was analyzed by size exclusion chromatography in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl to assess protein aggregation or degradation. Isocratic elution from a Superdex 200 Increase column was performed at 0.5 ml / min in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl.

[0074] Figure 6 shows the elution profile of truncated FGF-2 characterized by SEQ ID NO: 3 on Superdex200 Increase 10 / 300 after 30 days of storage at 4°C. The lower line is absorbance at 260 nm and the upper line is absorbance at 280 nm. The straight line above the absorbance at 280 nm is the system pressure and the upper line is conductivity.

[0075] The thermostabilized truncated FGF-2 corresponding to SEQ ID NO:3 eluted from the Superdex200 Increase 10 / 300 column as the major peak. The elution volume was 19 mL, similar to the elution time observed on the first day of purification. No shift to higher molecular weight was observed. Two minor peaks were detected at elution volumes of 20.1 mL and 21.1 mL, which are likely degradation products. Peak integration analysis indicates that the two minor peaks reflect 38% of the total FGF-2.

[0076] Long-term stability analysis showed that 38% of the FGF-2 was degraded after 30 days of storage at 4°C.

[0077] Example 5: Stability analysis after freeze-thaw cycles A stability analysis of the thermostabilized truncated FGF-2 characterized by SEQ ID NO:3 after one freeze-thaw cycle was performed.

[0078] FGF-2 (1 mg / ml) in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl was frozen in liquid nitrogen. After 1 hour, the protein was thawed and loaded onto a Superdex 200 Increase 10 / 300 column to examine protein aggregation or degradation. Isocratic elution from the Superdex 200 Increase column was performed at 0.5 mL / min in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl.

[0079] Figure 7 shows the elution profile of thermostabilized truncated FGF-2 corresponding to SEQ ID NO: 3 on a Superdex200 Increase 10 / 300 column after freezing and thawing. The bottom line is the absorbance at 260 nm (lower main peak), and the line above it is (higher main peak). The line above the absorbance at 280 nm is the system pressure, and the top line is the conductivity.

[0080] FGF-2 eluted from Superdex 200 increased by 10 / 300 as the main peak. The elution volume was 19 mL, which was similar to the elution time observed on the first day of purification. No shift to higher molecular weight was observed. A minor peak was observed at an elution volume of 21.1 mL, which is likely a degradation product. Peak integration analysis indicated that the minor peak reflected 10% of the total FGF-2.

[0081] Approximately 10% degradation of the heat-stabilized truncated FGF-2 was observed after one freeze-thaw cycle.

[0082] Example 6: Biological activity test The biological effects of the FGF-2 polypeptide according to the present invention (thermostabilized truncated FGF-2, SEQ ID NO: 3) were tested and compared with commercially available FGF-2 (Figure 8). Three different concentrations of FGF-2 were tested: 100, 10, and 1 ng / ml.

[0083] In the case of commercially available FGF-2, the cell proliferation achieved generally decreased with decreasing FGF-2 concentration. In the case of thermostabilized truncated FGF-2 (SEQ ID NO: 3), the cell proliferation achieved was comparable when concentrations of 100 and 10 ng / ml were applied, but the 1 ng / ml concentration resulted in lower cell numbers.

[0084] The thermostabilized truncated FGF-2 (SEQ ID NO: 3) exhibited stronger biological effects 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, 1.5-fold, 2.8-fold, and 1.7-fold higher cell numbers were observed, respectively.

[0085] Experimental details for bioactivity testing: Cells: C2C12 (seeding density: 4,000 cells / well) Culture vessel: 24-well plate ● Basal medium: Essential 8 (trademark) basal medium FGF-2 variants tested: No FGF-2 Commercially available FGF-2 - concentrations of 1, 10 and 100 ng / ml Heat-stabilized truncated FGF-2 (SEQ ID NO: 3) - concentrations of 1, 10 and 100 ng / ml Culture time points: Day 0 - Seeding Day 3 - Cell counting Day 7 - Cell counting, end of experiment

[0086] Example 7: Preparation of other thermostabilized FGF-2 polypeptides according to the present invention (SEQ ID NOS: 5-27) Thermostabilized FGF-2 polypeptides according to the present invention corresponding to SEQ ID NOs: 5 to 27 were prepared according to the procedure described in Example 1.

[0087] 9 shows the results of 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 dimeric thermostable FGF-2: SEQ ID NO: 22, (9) cells expressing dimeric thermostable FGF-2: SEQ ID NO: 23. [Industrial Applicability]

[0088] According to the present invention, FGF-2 polypeptides with improved stability can be used in many industrial applications, including scientific research, biotechnology research, medicine, pharmaceuticals, cosmetics, the clean meat industry, organoid generation, 3D cell culture models, and other related applications.

[0089] Sequence Listing SEQ ID NO: 1 Homo sapiens FGF-2 MAAGSITTLPEDGGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS SEQ ID NO: 2 Bos taurus FGF-2 MAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYSSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 3 Cleaved thermostabilized FGF-2 FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO:4 Heat-stabilized FGF-2 MAAGSITTLPALPEDGGSGAFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO:5 FGF-2 polypeptide variant 5 FPPGHFKDPKILYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO:6 FGF-2 polypeptide variant 6 FPPGHFKDPKVLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO:7 FGF-2 polypeptide variant 7 FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPWIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO:8 FGF-2 polypeptide variant 8 FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPIIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO:9 FGF-2 polypeptide variant 9 FPPGHFKDPKILYCKNGGFFLRIHPDGRVDGTRDKSDPWIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 10 FGF-2 polypeptide variant 10 FPPGHFKDPKILYCKNGGFFLRIHPDGRVDGTRDKSDPIIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 11 FGF-2 polypeptide variant 11 FPPGHFKDPKVLYCKNGGFFLRIHPDGRVDGTRDKSDPWIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 12 FGF-2 polypeptide variant 12 FPPGHFKDPKVLYCKNGGFFLRIHPDGRVDGTRDKSDPIIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 13 FGF-2 polypeptide variant 13 MAAGSITTLPALPEDGGSGAFPPGHFKDPKILYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 14 FGF-2 polypeptide variant 14 MAAGSITTLPALPEDGGSGAFPPGHFKDPKVLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 15 FGF-2 polypeptide variant 15 MAAGSITTLPALPEDGGSGAFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPWIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 16 FGF-2 polypeptide variant 16 MAAGSITTLPALPEDGGSGAFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPIIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 17 FGF-2 polypeptide variant 17 MAAGSITTLPALPEDGGSGAFPPGHFKDPKILYCKNGGFFLRIHPDGRVDGTRDKSDPWIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 18 FGF-2 polypeptide variant 18 MAAGSITTLPALPEDGGSGAFPPGHFKDPKILYCKNGGFFLRIHPDGRVDGTRDKSDPIIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 19 FGF-2 polypeptide variant 19 MAAGSITTLPALPEDGGSGAFPPGHFKDPKVLYCKNGGFFLRIHPDGRVDGTRDKSDPWIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 20 FGF-2 polypeptide variant 20 MAAGSITTLPALPEDGGSGAFPPGHFKDPKVLYCKNGGFFLRIHPDGRVDGTRDKSDPIIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 21 FGF-2 polypeptide variant 21 FGF2_mut_trunc_L83Y FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYYAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 22 FGF-2 dimeric polypeptide variant 22 FGF2_mut_trunc_GSS6x FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKSGSSGSSGSS GSSGSSGSSFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 23 FGF-2 dimeric polypeptide variant 23 FGF2_mut_trunc_GSS10x FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKSGSSGSSGSSGSSGSS GSSGSSGSSGSSGSSFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 24 FGF-2 dimeric polypeptide variant 24 FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKSSITVRVRDQTGEETFFKIKKTTKMQKVFETYATRKG VQVNSLRFLLDGDRITPDQTPKMLELEDQDQIDCVLFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 25 FGF-2 dimeric polypeptide variant 25 FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKSGGGGSSSITVRVRDQTGEETFFKIKKTTKMQKVFETYATRKG VQVNSLRFLLDGDRITPDQTPKMLELEDQDQIDCVLGGGGSFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 26 FGF-2 dimeric polypeptide variant 26 FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKSPSPSPSSITVRVRDQTGEETFFKIKKTTKMQKVFETYATRKG VQVNSLRFLLDGDRITPDQTPKMLELEDQDQIDCVLPSPSPSFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 27 FGF-2 dimeric polypeptide variant 27 FPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKSMDVPATTEDVKESAESITVRVRDQTGEETFFKIKKTTKMQKVFETYATRKG VQVNSLRFLLDGDRITPDQTPKMLELEDQDQIDCVLEQTGGKGHGQIGAAAQFPPGHFKDPKLLYCKNGGFFLRIHPDGRVDGTRDKSDPFIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLYAIKNVTDECFFFERLEENNYNTYRSRKYPSWYVALKRTGQYKLGPKTGPGQKAILFLPMSAKS SEQ ID NO: 28 SUMO Short SITVRVRDQTGEETFFKIKKTTKMQKVFETYATRKGVQVNSLRFLLDGDRITPDQTPKMLELEDQDQIDCVL SEQ ID NO: 29 SUMO short fl GGGGSSSITVRVRDQTGEETFFKIKKTTKMQKVFETYATRKGVQVNSLRFLLDGDRITPDQTPKMLELEDQDQIDCVLGGGGS SEQ ID NO: 30 SUMO Short RL PSPSPSSITVRVRDQTGEETFFKIKKTTKMQKVFETYATRKGVQVNSLRFLLDGDRITPDQTPKMLELEDQDQIDCVLPSPSPS SEQ ID NO: 31 SUMO Chief MDVPATTEDVKESAESITVRVRDQTGEETFFKIKKTTKMQKVFETYATRKGVQVNSLRFLLDGDRITPDQTPKMLELEDQDQIDCVLEQTGGKGHGQIGAAAQ

Claims

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

2. A thermostable FGF-2 polypeptide derived from Bos taurus FGF-2 polypeptide (SEQ ID NO: 2), characterized by deletion of amino acids 1-15 through 1-22 from the N-terminus.

3. 3. The thermostable FGF-2 polypeptide of claim 2, which is derived from the Bos taurus FGF-2 polypeptide (SEQ ID NO: 2), characterized by a deletion of amino acids 1 to 20 at the N-terminus.

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

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

3.

6. 6. The thermostable FGF-2 polypeptide of claim 5, having at least 93% sequence identity with SEQ ID NO:

3.

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

3.

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

3.

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

3.

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

4.

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

4.

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

4.

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

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

4.

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

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

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

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

3.

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

27.

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

21. 21. The thermostable FGF-2 polypeptide of claim 20, wherein the melting temperature (Tm) is 65°C or higher.

22. 21. The thermostable FGF-2 polypeptide of claim 20, wherein the melting temperature (Tm) is 68°C or higher.

23. Use of a thermostable FGF-2 polypeptide according to any one of claims 1 to 22 in the preparation of a cultured meat product.

24. 24. Use of the thermostable FGF-2 polypeptide of claim 23 in the preparation of a cultured meat product, wherein the cell culture process is carried out in a cell culture system 1 comprising at least one of a cell culture device 2, a cell harvesting device 3, a central control unit 4, or a monitoring device 5.

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

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

27. 27. The culture medium of claim 26, comprising a thermostable FGF-2 polypeptide characterized by SEQ ID NOs: 3-27.

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