Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/50—Fibroblast growth factor [FGF]
  • C07K14/503—Fibroblast growth factor [FGF] basic FGF [bFGF]

Definitions

• the invention relates to Fibroblast Growth Factor 2 (FGF-2) polypeptide with improved stability, especially thermal stability, compared to the wild-type of FGF-2 and the use thereof in research and industrial applications, such as biotechnological research, medicine, pharmaceutical industry, cosmetics, clean meat industry, generation of organoids and 3D cell culture models and other related applications.
• FGFs Fibroblast growth factors
• FGFs are a family of cell signaling proteins. They are involved in a variety of processes, especially as key elements for normal development in animal cells.
• FGF receptors 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 cell processes, such as proliferation, differentiation and survival, which make FGF signaling susceptible to subversion by cancer cells. FGFs are secreted glycoproteins that are generally readily sequestered to the extracellular matrix, as well as the cell surface, by heparan sulphate proteoglycans (HPSGs).
• HPSGs heparan sulphate proteoglycans
• FGFs are released from the extracellular matrix by heparinases, proteases or specific FGF-binding proteins, and the liberated FGFs subsequently bind to cell surface HPSGs.
• Cell surface HPSGs also stabilize the FGF ligand–receptor interaction, forming a ternary complex with FGFR.
• FGF receptors signal as dimers, and ligand-dependent dimerization leads to a conformational shift in receptor structure that activates the intracellular kinase domain, resulting in intermolecular transphosphorylation of the tyrosine kinase domains and intracellular tail.
• the human FGF-2 gene encodes not one protein, but a complex set of isoforms.
• the secreted isoform is a single-chain, non-glycosylated polypeptide with 154 amino acids.
• the amino acid sequence of human FGF-2 is 99% homologous to that of bovine FGF-2 and has high homology with ovine and rodent FGF-2, suggesting strong sequence conservation for structure and function.
• the stability of FGF-2 is widely accepted to be a major concern in the development of useful medicinal products or serum replacement in biotechnological research.
• the reconstituted FGFs solutions are stable for only about a week at 4 °C and are recommended to be used within 24 h when at ambient temperatures (around 25 °C). Nevertheless, a 50% loss of functionality of FGF-2 solutions at a concentration of 72 ⁇ g/mL was observed after just 4 min at 25 °C.
• One of the challenges is to preserve the biological activity of FGF-2.
• FGF-2 farnesoid GF-2
• approaches to maintain the stability and biological activity of FGF-2 include modulating ionic interactions in solution and chemical modifications of FGF-2.
• excipients to aqueous solutions of FGF-2 is among the simplest methods for FGF-2 stabilization by way of modulating ionic interactions in solution.
• Common strategies include the complexation of FGF-2 with its endogenous stabilizer, heparin or heparin-like polymers, or polycations. Ionic interactions between FGF-2 and the additives reduce the structural energy at the heparin-binding site, stabilize the FGF-2 native conformation and prolong its bioactivity in aqueous media.
• Mutant variants of the FGF-2 protein have been developed by aligning the wild-type FGF-2 protein sequence with stabilized FGF-1 mutant sequences, or by combining several individual stabilizing mutations identified by other investigators in the field. The identification of useful point mutations has been aided by computer modeling. Sequences have been identified that are important for protein functionality and contribute the greatest amount to structural free energy. Following an analysis of sequence conservation, mutations in regions, which might compromise protein function, were avoided. Conversely, mutations that were likely to result in a decrease in protein free energy were promoted. The obtained FGF-2 mutants showed the lowest energy and had increased functional half-life. Despite some advances within the state of the art, there is still need to further improve the stability of the FGF-2 product while maintaining its biological activity.
• the object of the invention is to prepare an FGF-2 variant with improved stability and to find an efficient, scalable and economically advantageous process for the production of FGF-2 with high yields.
• the disadvantages of the solutions, according to the state of the art, are solved by the present invention that provides a thermostable polypeptide that possesses FGF-2 activity and has increased stability while maintaining biological activity compared to the wild-type FGF-2.
• the product is truncated thermostabilized FGF-2 polypeptide with amino acid substitutions characterized by sequence SEQ ID NO: 3.
• the FGF-2 polypeptide with 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).
• the product is a thermostabilized FGF-2 polypeptide with amino acid substitutions characterized by sequence SEQ ID NO: 4.
• the objects of the present invention are also other FGF-2 polypeptides that are 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 thermostabilized FGF-2 polypeptide (SEQ ID NO: 3), FGF-2 polypeptides characterized by SEQ ID NO: 5-12 or SEQ ID NO:21 (which contains substitution L63Y), or other FGF-2 polypeptides according to the description below.
• 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 thermostabilized FGF-2 polypeptide (SEQ ID NO: 3), FGF-2 polypeptides characterized by SEQ ID NO: 5-12 or SEQ ID NO:21 (which contains substitution L63Y), or other FGF-2 polypeptides according to the description below.
• the objects of the present invention are also other FGF-2 polypeptides that are 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 thermostabilized FGF-2 polypeptide (SEQ ID NO: 4), for example FGF-2 polypeptides characterized by SEQ ID NO: 13 to 20.
• the objects of the present invention are also dimeric variants of the FGF-2 polypeptide comprising a linker, for example GSS linker or SUMO linker.
• the objects of the present invention are also dimeric variants of the FGF-2 polypeptide characterized by SEQ ID NO: 22-27.
• the dimeric variants of the FGF-2 polypeptide may comprise two connected sequences characterized by SEQ ID NO: 3 or two connected sequences having at least 90%, or 93%, or 95% sequence identity to the SEQ ID NO: 3.
• the linker for connecting 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 NO: 22 – 23, or SUMO linkers characterized by SEQ ID NO: 28-31 as used in dimeric FGF-2 polypeptides characterized by SEQ ID NO: 24 - 27.
• the invention also provides the process for preparation of the FGF-2 polypeptides and the use thereof in the pharmaceutical industry, cosmetics, clean meat industry generation of organoids and 3D cell culture models and other related applications.
• the increased stability while maintaining biological activity compared to the Homo sapiens FGF-2 has been proven by experiments.
• the biological activity has been tested by cell growth experiments, the biological effects of commercially available FGF-2 and FGF-2 products according to the invention have been compared.
• the thermal stability of the FGF-2 polypeptides using nano differential scanning fluorimetry (nanoDSF) method, and the effect of long-term storage on product stability have been investigated. Brief description of the several views of the drawings Fig.
• Fig.5 Thermostability of truncated thermostabilized 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 330/350 nm was plotted against the temperature. Representative thermal unfolding curve (top) and its first derivative analysis (bottom) Fig. 6 - Stability of truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 after storage at 4°C for 30 days Fig.
• thermostabilized FGF-2 characterized by SEQ ID NO: 3 after freezing-thawing cycle
• Fig. 8 The results from the cultivation of cells, when commercially available FGF-2 and truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 were compared
• Fig. 9 SDS-PAGE analysis of the expression of thermostabilized FGF-2 polypeptides.
• thermostable polypeptides that possess FGF-2 activity and have increased stability compared to the wild-type FGF-2, while maintaining the biological activity.
• the invention also provides the process for preparation of the FGF-2 polypeptides and use thereof.
• the product is a thermostable FGF-2 polypeptide derived from Bos taurus FGF-2 (SEQ ID NO: 2) comprising at least one of the amino acids substitutions R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, S121P.
• the product is a truncated thermostabilized FGF-2 polypeptide characterized by sequence SEQ ID NO: 3, Fig. 1.
• the sequence 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 nine amino acids substitutions, specifically R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, S121P.
• the Bos taurus FGF-2 SEQ ID NO: 2
• the truncated thermostabilized FGF-2 polypeptide SEQ ID NO: 3 have the modification S137P.
• the FGF-2 polypeptides may be derived from the Bos taurus FGF-2 polypeptide (SEQ ID NO: 2), with deletion of amino acids 1-15 to 1-22 at the N- terminus. These polypeptides may comprise at least one of amino acids substitutions: R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, or S121P relative to SEQ ID NO: 2.
• thermostabilized FGF-2 polypeptide (SEQ ID NO: 3), according to 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).
• the product is a thermostabilized FGF-2 polypeptide characterized by sequence SEQ ID NO: 4, Fig. 1. Nucleotide sequence encoding the thermostabilized FGF-2 was cloned into the pET30a(+) expression vector.
• thermostabilized FGF-2 polypeptide (SEQ ID NO: 4), according to the present invention, has 93.5% sequence identity to Homo sapiens FGF-2 a sequence (SEQ ID NO: 1) and 94.2% sequence identity to Bos taurus FGF-2 (a sequence SEQ ID NO: 2).
• the objects of the present invention are also other FGF-2 polypeptides that are 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 thermostabilized FGF-2 polypeptide (SEQ ID NO: 3).
• the objects of the present invention are also FGF-2 polypeptides derived from the amino acid sequence of SEQ ID NO: 3 comprising at least one of amino acids substitutions: R11L, V32T, E34D, H39F, L72Y, S74I, C76N, S89E, or S101P.
• the objects of the present invention are also FGF-2 polypeptides characterized by SEQ ID NO: 5-12 or SEQ ID NO: 21 (which contains substitution L63Y), or other FGF-2 polypeptides according to the description below.
• the objects of the present invention are also other FGF-2 polypeptides that are 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 thermostabilized FGF-2 polypeptide (SEQ ID NO: 4), for example FGF- 2 polypeptides characterized by SEQ ID NO: 13 to 20.
• the thermostable FGF-2 polypeptide derived from the amino acid sequence of SEQ ID NO: 4 may comprise at least one of the amino acids substitutions R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, or S121P.
• the products are dimeric variants of the FGF-2 polypeptide comprising a linker, for example GSS linker or SUMO linker.
• the objects of the present invention are also dimeric variants of the FGF-2 polypeptide characterized by SEQ ID NO: 22-27.
• the dimeric variants of the FGF-2 polypeptide according to the invention may comprise two connected sequences characterized by SEQ ID NO: 3 or two connected sequences having at least 90%, or 93%, or 95% sequence identity to the SEQ ID NO: 3.
• the linker for connecting two sequences may be for example a GSS linker (6xGSS or 10xGSS) as used in dimeric FGF-2 polypeptides characterized by SEQ ID NO: 22 – 23, or SUMO linkers characterized by SEQ ID NO: 28-31 as used in dimeric FGF-2 polypeptides characterized by SEQ ID NO: 24 - 27.
• the thermostabilized FGF-2 polypeptide may comprise amino acid substitution R31L (arginine at position 31 substituted by leucine) and H59F (histidine at position 59 substituted by phenylalanine) in the amino acid sequence of the Bos taurus FGF- 2 polypeptide (SEQ ID NO: 2).
• thermo-stabilized FGF-2 polypeptide may comprise another amino acid substitutions than R31L and H59F substitution in SEQ ID NO: 2 or SEQ ID NO: 4.
• thermostabilized FGF-2 polypeptide may comprise R31 substitution with any appropriate amino acid, for example with isoleucine or valine, and H59 substitution with any appropriate amino acid, for example with tryptophan or isoleucine.
• the object of the present invention is also FGF-2 polypeptides (SEQ ID NO: 5-12) that are derived from the amino acid sequence of SEQ ID NO: 3 and comprising 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).
• the object of the present invention is also FGF-2 polypeptides (SEQ ID NO: 13-20) that are derived from the amino acid sequence of SEQ ID NO: 4 and comprising 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).
• the object of the present invention is also thermostabilized FGF-2 polypeptides with the 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.
• thermostabilized FGF-2 polypeptides 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.
• the process according to the invention may consist of the steps: - Transformation of the competent E. coli cells with the plasmid DNA carrying the relevant FGF-2 sequence - Selection and screening of the transformed cells - Cultivation of the transformed cells - Induction of FGF-2 production - FGF-2 detection - FGF-2 isolation and purification - FGF-2 characterization.
• the bacterial expression host system for example E.
• coli bacterial expression host system may be used for the production of FGF-2 polypeptides due to its low cost, well-known biochemistry and genetics, rapid growth, and good productivity.
• a bacterial expression system is ideal for FGF-2 production as FGF-2 is a relatively small and single-domain protein with a compact fold.
• crystal structures of FGF-FGFR-heparin complexes and molecular mechanisms of the action of FGF proteins suggest that no post-translational modifications or cofactors of FGF proteins are required for their proper function.
• FGF proteins purified from E. coli showed biological activity. Therefore, the bacterial expression host system appears to be ideal for the large-scale industrial production of FGF-2. The presence of rare codons may be addressed by using codon optimization.
• the strains used for the production of FGF-2 polypeptides according to the invention may be E. coli strains, for example, BL21, BL-21-Gold or BL21- CodonPlus RIPL.
• the competent cells for the production of FGF-2 polypeptides are transformed with the plasmid DNA carrying the relevant FGF-2 sequence, encoding for example the FGF- 2 polypeptide as defined by sequence SEQ ID NO: 3 or SEQ ID NO: 4, or any other sequences according to the invention, e.g. SEQ ID NO: 5 to 27.
• the transformed cells are plated onto solid selection media, for example suspension of cells may be plated on LB (Luria-Bertani) agar plates supplemented with antibiotics.
• the colonies are later, for example next day, picked and transferred into liquid selection media, for example LB broth supplemented with antibiotics.
• liquid selection media for example LB broth supplemented with antibiotics.
• the cells are grown for several hours, for example overnight.
• the culture may be then placed into fresh liquid selection medium, for example LB broth supplemented with antibiotics, and incubated at temperature in the range of 15 to 45 °C, or in the range of 20 to 40 °C, or in the range of 30 to 37 °C.
• an antibiotic may be used for example kanamycin, or any other appropriate antibiotic.
• an inducing agent for example isopropyl ⁇ -D-thiogalactoside (IPTG), or any other appropriate inducing agent may be added to cell cultures for induction of FGF-2 production.
• IPTG isopropyl ⁇ -D-thiogalactoside
• the concentration of IPTG may be in the range of 0.001 to 10 mM, or in the range of 0.01 to 1 mM, or in the range of 0.05 to 0.5 mM.
• the cell cultures are incubated at a temperature in the range of 4 to 40 °C, or in the range of 15 to 30 °C, or in the range of 18 to 25°C.
• the culture may be harvested, for example by centrifuging at 13000 g, for example at room temperature.
• the cell pellets may be resuspended in 150 ⁇ l of 1x lithium dodecyl sulphate (LDS) gel sample buffer and heated to the temperature, which may be in the range of 60 to 100 °C, or in the range of 80 to 98 °C, or in the range of 90 to 95 °C for the time period, which may be in the range of 1 to 60 minutes, or in the range of 2 to 30 minutes, or in the range of 3 to 10 minutes.
• LDS lithium dodecyl sulphate
• the samples may be spun down at 13000 g, for example at room temperature, and analyzed with for example gradient SDS-PAGE.
• the volume of the sample analyzed may be in the range of 1 ⁇ l to 15 ⁇ l, or in the range of 2 to 12 ⁇ l, or in the range of 5 to 10 ⁇ l.
• the purification of the FGF-2 polypeptide according to the invention may be carried out for example by the following procedure. Post-induction bacterial cells from one liter cultivation are harvested, for example by centrifugation at 5000 g for 20 minutes. The cell pellet is lysed, for example by addition of lysozyme and by sonication.
• the lysate is clarified by centrifugation, for example at 75000 g at 4 °C for 30 minutes, and filtered through a 0.22 ⁇ m membrane.
• the clarified lysate is loaded onto a HisTrap column.
• the FGF-2 polypeptide is eluted by imidazole and purified with cation exchange chromatography, for example using HiTrap SP Sepharose.
• the FGF-2 polypeptide may be analyzed by gel filtration chromatography, using for example Superdex200 Increase column (10/300).
• the thermostability of the FGF-2 polypeptide may be analyzed, for example by nanoDSF.
• the long-term stability of the FGF-2 polypeptides according to the invention may be performed.
• the FGF-2 polypeptide may be stored in a fridge, e.g. at 4 °C for 30 days, and the protein may be then analyzed by size-exclusion chromatography
• the stability analysis after one or more freeze-thaw cycles of the FGF-2 polypeptides according to the invention may be performed. For example by the process described in example 5.
• the biological activity of the FGF-2 polypeptides according to the invention may be tested and the achieved cell growth may be determined.
• the different concentrations of the FGF-2s may be tested, for example the concentration of FGF-2 may be 100, 10 or 1 ng/ml.
• the thermostabilized truncated FGF-2 (SEQ ID NO: 3) had a stronger biological effect than the commercial FGF-2.
• the melting temperature (Tm) was determined, and the effect of the mutations (deletion at the N-terminus and nine substitutions in the FGF-2 chain) to increase FGF-2 stability was evaluated.
• the FGF-2 polypeptides have been prepared in high yields.
• the products showed excellent thermal stability and very good long-term stability at 4°C.
• the products may be frozen and successfully recovered.
• the FGF-2 polypeptides according to the invention may be successfully used in biotechnological research and industrial applications, medicine, the pharmaceutical industry, cosmetics, clean meat industry, generation of organoids and 3D cell culture models and other related applications.
• the FGF-2 polypeptides according to the invention may be used in many biotechnological processes, for example for cell cultivation or for biotechnological production of many different types of requested compounds, such as for example proteins, active pharmaceutical ingredients or antibodies.
• the FGF-2 polypeptides according to the invention may be used in cell cultivation processes for the purpose of preparing the cultured meat products for human consumption or as a pet food.
• the FGF-2 polypeptides according to the invention may be used as a component of the culture media. These processes of cell cultivation may be carried out in a cell cultivation system 1, as depicted on Fig. 10.
• the cell cultivation system 1 may comprise at least one of: a cultivation device 2, formed for example by a production bioreactor, a cell harvesting device 3, a central control unit 4, or a monitoring device 5.
• the cultivation device 2, e.g. the production bioreactor, the cell harvesting device 3, the central control unit 4, and/or the monitoring device 5 may be in direct or indirect connection and/or communication with each other.
• the cell harvesting device 3 may comprise a filtration device, a centrifugation device, or any other appropriate device for harvesting of cells.
• the system may further comprise for example a seeding tank or a device for preparing cultured meat composition (not depicted on Fig.10).
• thermostable FGF-2 polypeptides according to the invention may be used as a component of the culture medium in may biotechnological processes, for example in cultivation of mammalian cells, for example for the purpose of preparing cultured meat products in clean meat industry.
• the thermostable FGF-2 polypeptides according to the invention may be used as signaling compounds in the culture medium.
• the culture medium according to the invention may further comprise amino acids or their sources, in combination with at least one type of compounds that may be selected from a group comprising: sugars, fatty acids, vitamins and organic micronutrients, mineral compounds, supplements, such as for example iron supplementation compounds, organic amines, shear protectants, additional compounds, or any other appropriate compounds.
• Example 1 preparation of the truncated thermostabilized FGF-2 polypeptide (SEQ ID NO: 3) The competent cells of three selected E. coli strains (BL21, BL-21-Gold and BL21-CodonPlus RIPL) were transformed with the plasmid DNA of the thermostabilized truncated construct FGF-2 corresponding to SEQ ID NO. 3, and each cell suspension was plated on agar plates supplemented with kanamycin. The next day, two colonies from each plate were picked into 5 mL LB broth supplemented with kanamycin and grown overnight.
• SEQ ID NO: 3 The competent cells of three selected E. coli strains (BL21, BL-21-Gold and BL21-CodonPlus RIPL) were transformed with the plasmid DNA of the thermostabilized truncated construct FGF-2 corresponding to SEQ ID NO. 3, and each cell suspension was plated on agar plates supplemented with kanamycin. The next day, two colonies from each plate were picked into 5 mL
• Fig.2 depicts the SDS-PAGE analysis of the expression of truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 in E. coli BL21 strain. The expression was induced by the addition of 1 mM IPTG and was performed at 20°C for 1, 3, 5, 8, 12, 18 and 24 h. The truncated thermostabilized FGF-2 (SEQ ID NO: 3) was successfully expressed in all three E.
• LDS lithium dodecyl sulphate
• Example 2 preparation of the truncated thermostabilized FGF-2 polypeptide (SEQ ID NO: 3) and the purification thereof on HisTrap FF column and Superdex200 Increase column
• the BL21 (DE3) competent cells were transformed with the plasmid DNA of truncated construct FGF-2 and plated on agar plates supplemented with kanamycin.
• 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 (5 mg in total), and lysozyme (100 mg in total). The suspension was incubated on ice for 30 minutes.
• the cells were lysed by sonication on ice for 8 min (10s On/20s Off, amplitude 40%), and the lysate was clarified by centrifugation at 75 000 g at 4 °C for 30 minutes.
• the lysate was filtered through a 0.22 ⁇ m membrane and loaded at a 5 mL/min flow rate onto the HisTrap FF (5 mL) column that was equilibrated in 30 mM HEPES (pH 7.5), 500 mM NaCl and 10 mM imidazole.
• the 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 VivaSpin Turbo centrifugal concentrators with 10 kDa cut-off and diluted 20 x with 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 minutes, and the gradient was set from 0 to 60 % of the 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 VivaSpin Turbo centrifugal concentrators with 10 kDa cut-off and loaded onto a Superdex200 Increase column (10/300).
• the 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.
• the results of purification of the truncated FGF-2 characterized by SEQ ID NO: 3 were controlled using SDS PAGE analysis (Fig. 3). After sonication, the lysate was centrifuged, and 10 ⁇ l of supernatant (1) and pellet (2) were loaded on the gel.
• the supernatant was then loaded onto a HisTrap column and flow through (3), fraction after washing with buffer containing 10 mM imidazole (4), 40 mM imidazole (5), and finally with 150 mM imidazole (6) were loaded on the gel and analyzed using SDS PAGE.
• the elution profile of the truncated thermostabilized FGF-2 characterized by SEQ ID NO: 3 on the Superdex200 Increase 10/300 is shown in Fig. 4.
• the line with peak maximum at approximately 20.9 min represents conductivity.
• the upper line with peak maximum at approximately 17.6 min is absorbance at 280 nm
• the middle line is absorbance at 260 nm
• the bottom line is absorbance at 450 nm.
• thermostability assay The thermostability assay of FGF-2 was performed using nanoDSF in 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. The measurement was performed in triplicates at temperatures from 20 °C to 90 °C with a temperature ramp of 1.5 °C/min. Changes in tryptophan emission at 330 and 350 nm were monitored, and the ratio 330/350 nm was plotted against the temperature.
• thermostabilized truncated FGF-2 corresponding to the SEQ ID NO: 3 determined by nanoDSF was 68.4 °C (Fig. 5). Representative thermal unfolding curve (top) and its first derivative analysis, indicating the melting temperature of the protein (bottom) are shown.
• the experimental Tm was about 14.9 °C higher than the Homo sapiens FGF-2 (SEQ ID NO: 1). This value indicates excellent thermal stability, which is in agreement with the contributions of individual substitutions.
• Example 4 long term stability
• the thermo-stabilized truncated FGF-2 corresponding to the SEQ ID NO: 3 was stored in a fridge at 4 °C for 30 days postproduction, and the protein was analyzed by size-exclusion chromatography in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl in order to examine protein aggregation or degradation.
• the 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.
• FIG. 6 depicts the elution profile of the truncated FGF-2 characterized by SEQ ID NO: 3 on Superdex200 Increase 10/300 after 30 days of storage at 4 °C.
• the bottom line is absorbance at 260 nm, the line above that is absorbance at 280 nm.
• the straight line above the absorbance at 280 nm is the system pressure, and the upper line shows conductivity.
• the thermostabilized truncated FGF-2 corresponding to the SEQ ID NO: 3 was eluted from Superdex200 Increase 10/300 column as the main peak.
• the elution volume was 19 mL and was comparable to the elution time observed on the first day of purification. No shift toward higher molecular weights was observed.
• Example 5 stability analysis after freeze-thaw cycle The stability analysis after one freeze-thaw cycle of the thermostabilized truncated FGF-2 characterized by SEQ ID NO: 3 was performed. The FGF-2 (1 mg/ml) in a buffer containing 15 mM Hepes (pH 7.5) and 150 mM NaCl was frozen in liquid nitrogen.
• Fig. 7 depicts the elution profile of the thermostabilized truncated FGF-2 corresponding to the SEQ ID NO: 3 on the Superdex200 Increase 10/300 column after freezing and thawing.
• the bottom line is the absorbance at 260 nm (lower main peak), the line above that is (higher main peak).
• the straight line above the absorbance at 280 nm is the system pressure and the upper line shows conductivity.
• FGF-2 eluted from the Superdex200 Increase 10/300 as the main peak.
• the elution volume was 19 mL and was comparable to the elution time observed on the first day of purification. No shift toward higher molecular weights was observed.
• a minor peak was observed at an elution volume of 21.1 mL and is probably a degradation product. Peak integration analysis indicated that the minor peak reflects 10% of the total FGF-2. About a 10% degradation of the thermostabilized truncated FGF-2 was observed after one freeze-thaw cycle.
• thermostabilized truncated FGF-2 (SEQ ID NO: 3) had a stronger biological effect than the commercial FGF-2. For example, 1.5 x, 2.8 x and 1.7 x higher cell count has been observed in the case of the 100 ng/ml, 10 ng/ml and 1 ng/ml concentrations of the prepared FGF-2 polypeptide, respectively.
• thermostabilized FGF-2 polypeptides according to the invention SEQ ID NO: 5 – 27
• the thermostabilized FGF-2 polypeptides according to the invention corresponding to SEQ ID NO: 5 – 27 have been prepared according to the procedure described in example 1.
• thermostabilized FGF-2 polypeptides depicts SDS-PAGE analysis of the expression of thermostabilized FGF-2 polypeptides.
• (1) non-induced cells (2) control - cells expressing the truncated thermostabilized FGF2 – SEQ ID NO: 3, (3) cells expressing dimeric thermostabilized FGF-2 - SEQ ID NO: 24, (4) cells expressing dimeric thermostabilized FGF-2 SEQ ID NO: 25, (5) cells expressing dimeric thermostabilized FGF-2 - SEQ ID NO: 26, (6) cells expressing dimeric thermostabilized FGF- 2 - SEQ ID NO: 27, (7) cells expressing thermostabilized FGF-2 - SEQ ID NO: 21, (8) cells expressing dimeric thermostabilized FGF-2 - SEQ ID NO: 22, (9) - cells expressing dimeric thermostabilized FGF-2 - SEQ ID NO: 23.
• the FGF-2 polypeptides with improved stability may be used in scientific research as well as in many industrial applications, for example, in biotechnological research, medicine, pharmaceutical industry, cosmetics, clean meat industry generation of organoids and 3D cell culture models and other related applications.

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