Thermally stable fgf2 polypeptides, uses thereof

Abstract

The present invention provides isolated heat-stable polypeptides having FGF2 activity and having at least 85% sequence identity to SEQ ID NO: 2 (FGF2wt) or a functional fragment thereof and comprising at least one amino acid substitution R31L and uses thereof in cell biology research, regenerative medicine and related medical applications or cosmetics. Furthermore, disclosed are culture media comprising the subject FGF2 suitable for culturing human pluripotent stem cells involving both human embryonic stem cells and induced pluripotent stem cells.

Description

Invention Field

[0001] This invention relates to engineered fibroblast growth factor 2 (FGF2, bFGF) with improved thermal stability compared to the wild type, and its use in cell biology research, regenerative medicine and related medical applications or cosmetics. The invention also relates to a culture medium containing FGF2 suitable for culturing human pluripotent stem cells involving both human embryonic stem cells and induced pluripotent stem cells. Background of the Invention

[0003] Fibroblast growth factor 2 (FGF2, also known as basic FGF, bFGF) is a pleiotropic regulator of proliferation, differentiation, migration, and survival in various cell types, and is also a fundamental component of the culture medium used for human pluripotent stem cells (PSCs) because it helps maintain cells in a pluripotent state. Pluripotency is the ability of a cell to undergo unlimited self-renewal and differentiate into all cell types in the human body. This property makes cells valuable for studying embryogenesis, drug discovery, and cell-based therapies. Other important biological activities of FGF2 that encompass medical applications include promoting angiogenesis, promoting wound healing, promoting cartilage or bone formation, and promoting neurogenesis.

[0004] However, the low stability and short half-life of wild-type FGF2 are impractical for a variety of applications, including the culture of PSCs. The half-life of the wild-type molecule is less than 24 hours under conditions typically used for culturing human PSCs, necessitating frequent replacement, which is a cost concern in the industry (Lotz et al. 2013, PLoS One 8:e56289). Methods for culturing mammalian stem cells or progenitor cells in the presence of continuous concentrations of FGF2 are provided in patent document US 8,481,308. Furthermore, stem cells are exposed to fluctuations in FGF2 concentration due to continuous degradation, which can lead to a rapid reduction in appropriate signaling, crucial for pluripotency. Thermodynamic stability of proteins is particularly important in therapeutic applications, as unfolded or aggregated forms of proteins can have potential toxicity and immunogenicity.

[0005] Traditionally, FGF2 has been stabilized by adding heparin, which protects FGF2 from heat and acid denaturation and also prolongs its half-life. However, heparin is produced by mast cells in the body, so its use in most cells / tissues regulated by FGF2 in vivo is not physiological. Furthermore, the anticoagulant properties of heparin and the risk of inducing allergic reactions make such formulations less suitable for medical and cosmetic purposes. Therefore, there remains a need in the art for novel and improved methods that would allow for the acquisition of FGF2 compositions with greater stability and longer functional half-lives without requiring heparin at an affordable price.

[0006] Patent document WO2013 / 082196 describes conjugates of heparin-mimicking sulfonate (salt) polymers (such as poly(styrene sulfonate)) or copolymers (such as poly(styrene sulfonate-co-poly(polyethylene glycol methacrylate)) with FGF2 to stabilize FGF2 while retaining its full growth factor activity. Several patent documents, such as US 7,754,686 (addition of a reducing agent to inhibit FGF oxidation), US 5,202,311 (addition of sucrose octasulfate), US 5,189,148 (addition of water-insoluble hydroxypropyl cellulose), and EP0345660 (addition of dextran sulfate), describe stabilizing FGF by adding certain agents. However, as with the case of FGF2 formulated with heparin, a disadvantage of such formulations is the presence of potentially harmful compounds that are not suitable for medical and routine care purposes.

[0007] Protein engineering offers a powerful solution for stabilizing proteins without the need for additives. Therefore, mutants of FGF1 and FGF2, belonging to the same subfamily, with enhanced stability and / or function, are described. Compared to FGF2, FGF1 has even more limited biotechnological applications, primarily due to its inherently higher instability.

[0008] U.S. Patent Application No. 2008 / 038287 relates to the design, preparation, and use of FGF2 or FGF4 peptides with enhanced receptor specificity obtained by truncation and optional N-terminal amino acid substitution. However, they neither teach nor support that mutations or truncation in the N-terminal residues affect the thermal stability of FGF.

[0009] U.S. Patent Application No. 2012 / 0225479 relates to engineered human FGF2 mutants with increased thermal stability and methods for culturing embryonic stem cells using them. The authors employed substitutions Q65I, N111G, and C96S in the wild-type FGF2 sequence identified by a simple amino acid sequence comparison between FGF2 and stable FGF1 mutants reported by Zakrzewska et al. (Zakrzewska M, 2005 J Mol Biol). The described mutants exhibited a certain level of stabilization at higher temperatures but could not maintain their biological activity for extended periods.

[0010] U.S. Patent Application No. 2013 / 0236959 describes a specific thermostable FGF2 K128N mutant. K128 is an amino acid that, in the case of wild-type FGF2, significantly promotes the binding of heparin and heparan sulfate proteoglycan (HSPG). Therefore, since the binding of FGF2 to HSPG is one of the key functional components in FGF receptor activation, the amino acid substitution at this position reduces the ability of FGF2 to bind HSPG, which may negatively affect the specific biological activity of FGF2. The overall mechanism of FGF signaling involves heparin or HSPG, which act as co-receptors to promote FGF oligomerization and the binding of FGF to its tyrosine kinase receptor (FGFR), leading to FGFR oligomerization and signaling. Substitutions in the heparin / HSPG binding domain are also disclosed in U.S. Patent Application No. 2013 / 0157359. This application relates to two FGF1 variants with enhanced thermostability achieved by introducing three and four amino acid substitutions. Stabilization of heparin-independent FGF1 is achieved by mutating the residue K112, which is important for HSPG binding.

[0011] U.S. Patent No. 8,461,111 relates to an engineered FGF1 with an enhanced functional half-life achieved by introducing nuclear packaging mutations.

[0012] U.S. Patent No. 8,119,776 relates to engineered FGF1 with increased thermal stability and mitogenic efficacy by substituting residues 12 and 134.

[0013] Invention disclosure

[0014] The purpose of this invention is to provide a thermostable FGF2 that significantly reduces culture costs, leads to improved quality of cultured cells, and reduces operational requirements. Furthermore, it can be used in regenerative medicine and related medical applications, or in cosmetics.

[0015] This invention overcomes the shortcomings of existing technical solutions by presenting a separated, thermostable polypeptide with FGF2 activity and composed of an FGF2 polypeptide having 85% sequence identity with the sequence SEQ ID NO:2 or a fragment thereof. The FGF2 polypeptide also contains at least one amino acid substitution selected from R31L or H59F; or a combination of at least two substitutions for R31L and H59F. This means that the polypeptide according to the invention always exhibits at least one substitution for R31L or H59F; or a combination of at least two substitutions for R31L and H59F.

[0016] Advantageously, the FGF-2 peptide or fragment thereof described in the present invention exhibits bioactivity that remains stable and unchanged over long periods at high temperatures (see, for example, [link to article]). Figure 11 ).

[0017] The thermostable FGF2 peptides or fragments thereof according to the present invention particularly benefit from the fact that they are significantly more stable than wild-type FGF2. This stability is inherent to FGF2; no additional compounds, such as heparin, are required. This is true even if none of the amino acid positions essential to the biological activity of FGF2 are substituted or truncated. The described FGF2 mutants and their fragments can be used in clinical and research practices.

[0018] The thermostable FGF2 peptide or fragment thereof according to the present invention has FGF2 activity and a melting temperature that is increased by 1 to 20 °C, preferably 8 to 20 °C, and more preferably 14 to 20 °C compared to the wild-type FGF2 peptide. All 13 single-point mutants were constructed, subcloned into the expression vector pET28b, purified (purity ≥95%, as determined by SDS-PAGE analysis), and subsequently characterized for melting temperature.

[0019] In another aspect, the present invention provides an FGF2 polypeptide having at least 85% sequence identity with SEQ ID NO:2 or a fragment thereof and containing at least amino acid substitution R31L.

[0020] Preferred embodiments of the present invention disclose a thermostable FGF2 polypeptide having SEQ ID NO:2 or a fragment thereof comprising at least an amino acid substituted for R31L.

[0021] More preferably, the polypeptide comprises a sequence selected from the following sequences: SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26 or SEQ ID NO:28.

[0022] A more preferred embodiment of the present invention discloses a thermostable FGF2 polypeptide or a fragment thereof, wherein the basic substitution in the FGF2 polypeptide is R31L, and further comprises at least two, at least five, at least eight, or at least ten amino acid substitutions selected from the following: R31W, V52T, H59F, L92Y, C96Y, S109E, K30I, E54D, S94I, C96N, E108H, T121P.

[0023] A more preferred embodiment of the present invention discloses a thermostable FGF2 polypeptide or a fragment thereof, wherein the basic substitution in the FGF2 polypeptide is H59F, and further comprises at least two, at least five, at least eight, or at least ten amino acid substitutions selected from the following: R31W, R31L, V52T, L92Y, C96Y, S109E, K30I, E54D, S94I, C96N, E108H, T121P.

[0024] A more preferred embodiment of the present invention discloses a thermostable FGF2 polypeptide or a fragment thereof, wherein the basic substitution in the FGF2 polypeptide is a combination of R31L and H59F, and further comprises at least one, at least four, at least seven, or at least nine amino acid substitutions selected from the following: R31W, V52T, L92Y, C96Y, S109E, K30I, E54D, S94I, C96N, E108H, T121P.

[0025] A more preferred embodiment of the present invention discloses a polypeptide comprising: (a) a polypeptide having three amino acids replacing R31L, V52T, and H59F, most preferably having SEQ ID NO:30; or (b) a polypeptide having six amino acids replacing R31L, V52T, H59F, L92Y, C96Y, and S109E, most preferably having SEQ ID NO:32; or (c) a polypeptide having nine amino acids replacing K30I, R31L, V52T, E54D, H59F, L92Y, C96Y, E108H, and S109E, most preferably having SEQ ID NO:34; or (d) a polypeptide having nine amino acids replacing R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, and T121P, most preferably having SEQ ID NO:30. The polypeptide NO:36, and (e) eleven amino acid substitutions of K30I, R31L, V52T, E54D, H59F, L92Y, S94I, C96N, E108H, S109E, T121P, with the polypeptide having SEQ ID NO:38 being the most preferred.

[0026] The mutant proteins described below should also be considered part of the scope of this invention.

[0027] The bioactivity of the FGF2 polypeptide or fragment thereof or its mutant protein according to the present invention can be demonstrated by EC targeting the proliferation of NIH / 3T3 cells. 50 Quantitatively expressed, the range is 0.1 to 5 ng / mL, preferably 0.5 to 3 ng / mL. As previously described (Dubey et al. 2007 J Mol Biol), the bioactivity of FGF2 can be evaluated by a proliferation assay of cultured fibroblasts.

[0028] In a second aspect, the present invention provides a thermostable FGF2 polypeptide or fragment thereof according to the invention, which may be used in regenerative medicine (such as, for example, healing wounds and ulcers, fracture healing and periodontal tissue regeneration) and other medical applications (such as, for example, cancer treatment, cardiovascular disease therapy and mood disorder treatment) or cosmetics (such as, for example, hair stimulation, collagen synthesis support and anti-aging treatment).

[0029] Thirdly, the present invention provides a culture medium suitable for culturing undifferentiated human pluripotent stem cells, which contains an effective amount of the thermostable FGF2 polypeptide or a fragment thereof in the range of 1.0 ng / μl to 100 ng / μl of culture medium according to the present invention. Preferably, the culture medium comprises the FGF2 polypeptide or fragment thereof according to the invention containing the following amino acid substitutions: (a) R31L, V52T, H59F, most preferably a polypeptide having SEQ ID NO:30; or (b) R31L, V52T, H59F, L92Y, C96Y, S109E, most preferably a polypeptide having SEQ ID NO:32; or (c) K30I, R31L, V52T, E54D, H59F, L92Y, C96Y, E108H, S109E, most preferably a polypeptide having SEQ ID NO:34; or (d) R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, T121P, most preferably a polypeptide having SEQ ID NO:30. The polypeptide with SEQ ID NO:36, and (e)K30I, R31L, V52T, E54D, H59F, L92Y, S94I, C96N, E108H, S109E, T121P, are most preferably the polypeptide having SEQ ID NO:38.

[0030] These and other features, objects, and advantages of the invention will become more readily understood from the following description. In the description, reference is made to the accompanying drawings, which form part of the description and illustrate embodiments of the invention in an illustrative rather than limiting manner. Invention Details

[0032] definition

[0033] The following provides definitions for certain terms used in this specification. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] As used herein, the term 'thermostability' is synonymous with the protein term 'thermal stability' and encompasses both thermodynamic and kinetic stability. Thermodynamic stability relates to the equilibrium between a protein's folded (native) and unfolded states and is defined as the difference in Gibbs free energy between these two protein states.

[0035] As used in this article, the term 'melting temperature' (T) of FGF2 protein refers to... m Melting temperature refers to the temperature at which 50% of a protein folds and 50% of it unfolds. Melting temperature is a direct measure of thermodynamic stability.

[0036] As used herein, the term 'half-life' of FGF2 protein refers to the time it takes for the biological function of FGF2 protein to decrease by half under defined process conditions. For example, functional half-life can be based on the biological activity of FGF2 protein over time in inducing cell growth, proliferation, and / or survival. Half-life is a direct measure of kinetic stability, which is related to the energy barrier that separates the native state from the non-functional protein form (the unfolded state, an irreversibly denatured protein).

[0037] As used herein, the term 'wild-type' refers to naturally occurring FGF2 with the most common amino acid sequence in members of the species. In this context, wild-type FGF2 is human FGF2, which is an 18 kDa protein (SEQ ID NO:2) of 155 amino acids in length.

[0038] As used herein, the term 'FGF2 polypeptide' refers to a polypeptide having FGF2 activity, having at least 85% sequence identity with SEQ ID NO:2 or preferably having SEQ ID NO:2 and containing at least one amino acid substitution selected from R31L or H59F, or a combination of at least two substitutions of R31L and H59F, and having a Tg ratio compared to wild-type FGF2 protein. m Increase by at least 1°C, preferably at least 8°C, and more preferably at least 14°C. m It can be measured using any method suitable for determining melting temperature, such as circular dichroism spectroscopy, differential scanning calorimetry, and fluorescence thermal displacement measurement.

[0039] As used herein, the term '3-point FGF2 mutant' or 'FGF2 CS1' refers to an FGF2 polypeptide having SEQ ID NO:2 or a fragment thereof, comprising the following amino acid substitutions: R31L, V52T, H59F. Preferably, it is a polypeptide having SEQ ID NO:30. As used herein, the term '6-point FGF2 mutant' or 'FGF2 CS2' refers to an FGF2 polypeptide having SEQ ID NO:2 or a fragment thereof, comprising the following amino acid substitutions: R31L, V52T, H59F, L92Y, C96Y, S109E. Preferably, it is a polypeptide having SEQ ID NO:32.

[0040] As used herein, the term '9-point FGF2 mutant' or 'FGF2 CS3' refers to an FGF2 polypeptide having SEQ ID NO:2 or a fragment thereof, comprising the following amino acid substitutions: K30I, R31L, V52T, E54D, H59F, L92Y, C96Y, E108H, S109E. Preferably, it is a polypeptide having SEQ ID NO:34.

[0041] As used herein, the term '9-point FGF2 mutant' or 'FGF2 CS4' refers to an FGF2 polypeptide having SEQ ID NO:2 or a fragment thereof, comprising the following amino acid substitutions: R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, T121P. Preferably, it is a polypeptide having SEQ ID NO:36.

[0042] As used herein, the term '11-point FGF2 mutant' or 'FGF2 CS5' refers to an FGF2 polypeptide having SEQ ID NO:2 or a fragment thereof, or containing the following amino acid substitutions: K30I, R31L, V52T, E54D, H59F, L92Y, S94I, C96N, E108H, S109E, T121P. Preferably, it is a polypeptide having SEQ ID NO:38.

[0043] As used herein, the term 'FGF2 polypeptide' is synonymous with 'FGF2 mutant' and refers to a modified polypeptide sequence that, compared to the wild-type sequence FGF2 SEQ ID NO:2, has at least one different amino acid sequence exhibiting any of the substitutions according to the invention.

[0044] As used in this article, the term 'peptide' is synonymous with 'protein'.

[0045] As used herein, the term "FGF2 activity" is synonymous with the term "biological activity of FGF2." It refers to the biological activity of the FGF2 polypeptide or fragment thereof or mutant protein according to the invention. These retain the cell-binding portion and heparin-binding segment of the FGF2 protein described in the invention. They are capable of binding to at least one FGF receptor (FGFR) present on the cell surface, which is essential for transducing signals into the cell interior and inducing growth, proliferation, or survival of cultured cells relative to untreated control cells. Such cells may include, for example, mesenchymal-derived cells known in the art for expressing one or more FGFRs or responding to FGF proteins, typically fibroblasts, neuroblasts, glial cells and other neurally derived cells, smooth muscle cells, endothelial cells, etc. FGFRs include various isoforms of the receptor, including soluble forms containing an extracellular domain but lacking transmembrane and kinase domains. Biological activity can be measured by methods known in the art, such as cell proliferation and / or substrate phosphorylation.

[0046] As used herein, the term 'fragment' refers to a functional fragment of the FGF2 polypeptide according to the invention that has FGF2 activity. Furthermore, it refers to a functional fragment of the FGF2 polypeptide having at least 85% sequence identity with sequence SEQ ID NO:2. According to the invention, fragments of the FGF2 polypeptide also exhibit at least one or more substitutions. Preferably, at least 96%, 97%, 98%, 99%, or 100% sequence identity is present. This fragment means a polypeptide consisting only of a portion of the complete polypeptide sequence and structure, and may contain C-terminal or N-terminal deletions of this variant. Such functional fragments retain the cell-binding portion and heparin-binding segment of the FGF2 protein described in the invention. Compared to wild-type FGF2, fragments of the FGF2 protein described in the invention retain the desired properties, therefore their T... m The temperature is increased by at least 1°C, preferably at least 8°C, more preferably at least 14°C, and they are capable of binding to at least one FGF receptor present on the cell surface and inducing growth, proliferation or survival of the cultured cells relative to untreated control cells.

[0047] As used herein, the term 'mutant protein' refers to a functional mutant protein of the FGF2 protein or a fragment thereof according to the invention. Furthermore, it refers to a functional mutant protein of a polypeptide having at least 85% sequence identity with sequence SEQ ID NO:2, exhibiting any substitutions according to the invention. Preferably, at least 96%, 97%, 98%, 99%, or 100% sequence identity is desired. This means that they are mutant forms that retain any possible amino acid substitutions as described above for the FGF2 protein according to the invention and at least 85% or more of the residues of sequence SEQ ID NO:2. Such functional mutant proteins retain the biological activity of the FGF2 of the reference sequence. Preferably, the mutation is a substitution using L-amino acids, where one amino acid is replaced by another biologically similar amino acid. Examples of conserved substitutions include replacing one hydrophobic residue with another, or replacing one charged or polar residue with another. Preferably, the substitution is introduced at the N-terminus of FGF2, which is irrelevant to biological activity.

[0048] As used herein, the term 'sequence identity' means the presence of identical amino acid residues within the FGF2 protein according to the invention as defined above. The FGF2 protein is used as a reference when a specified contiguous segment of its amino acid sequence is aligned and compared with the amino acid sequence of a specific corresponding reference molecule. The percentage of sequence identity is calculated by the following steps: determining the number of positions in both sequences where identical amino acid residues occur to generate a number of matching positions; dividing the number of matching positions by the total number of positions in the segment compared with the reference molecule; and multiplying the result by 100 to obtain the percentage of sequence identity. Sequence alignment methods are well known in the art. The reference sequence used herein refers to the specific corresponding human FGF2 protein according to the invention. FGF2 is highly conserved in mammalian species such as mice, rats, rabbits, primates, pigs, dogs, cattle, horses, and humans, and exhibits at least 85% sequence identity across a wide range of species. Preferably, at least 96%, 97%, 98%, 99%, or 100% sequence identity is achieved. Those skilled in the art will understand that the remaining 15% or less of the amino acids along the length of the FGF2 protein according to the invention is variable, due to, for example, the use of FGF2 species from different sources or the addition of suitable non-FGF peptide sequences or tags known in the art. An FGF2 protein according to embodiments of the invention, having at least 85% identity with wild-type FGF2, cannot include proteins other than those similar to FGF2, because other members of the FGF family typically have much lower sequence identity.

[0049] As used in this article, the term 'effective amount' refers to the amount necessary to maintain pluripotent stem cells with an undifferentiated morphology for at least 5 generations.

[0050] As used herein, the term 'human pluripotent stem cells' refers to both human embryonic stem cells and induced pluripotent stem cells, characterized by their self-renewal capacity—the ability to form identical offspring of themselves—and the pluripotency that allows them to generate almost all cell types in the human body.

[0051] As used herein, the term 'maintaining stem cells in a pluripotent state' refers to maintaining cells in an undifferentiated state, possessing the ability to differentiate into almost all cell types. Pluripotency depends on a mixture of growth factors in which FGF2 is crucial for stemness-supporting. FGF2 supports self-renewal in several ways: it directly activates the mitogen-activated protein kinase pathway and indirectly promotes transforming growth factor β1 and activin signaling (Greber et al. 2008, Stem Cells 25, 455-464). Through its role in cell adhesion and survival, FGF2 promotes the pluripotency of human PSCs in a complex manner (Eisellova et al. 2009, Stem Cells 27, 1847-1857).

[0052] describe

[0053] The most attractive approach to overcoming the instability of FGF proteins is through mutagenesis to alter their properties. By changing their amino acid sequence, FGF proteins can potentially exhibit greater thermal stability, increased half-life, and increased resistance to proteolytic degradation. Even for human therapeutic applications, mutating proteins to optimize their properties is feasible. The FDA has approved several mutant forms of the protein for use as human drugs.

[0054] The disclosure of this invention provides an FGF2 polypeptide stabilized according to the invention through protein engineering. Stable mutations are rationally predicted through bioinformatics analysis and computational protein design. A hybrid approach combining information from evolutionary analysis and force field calculations is enriched through intelligent filtering and expert judgment. This method leads to highly reliable computer-predicted stable substitutions. Therefore, mutants are prepared by site-directed mutagenesis or screened from large saturated libraries using novel growth arrest assays. The final mutants are prepared by computational analysis of recombination and by gene synthesis or mutagenesis.

[0055] Typically, the gene encoding FGF2 is cloned and then expressed in a transformed organism, preferably a microorganism. The host organism expresses the foreign gene under expression conditions to produce FGF2. Synthetic recombinant FGF2 can also be prepared in eukaryotes such as yeast or human cells. FGF2 can be in a 146-amino acid form, a 153-155-amino acid form, or a mixture thereof, depending on the method of recombinant production (see U.S. Patent No. 5,143,829).

[0056] Melting temperature is a direct measure of thermodynamic stability. Examples of techniques used to measure melting temperature include circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC), and thermoluminescence shift assay (TSA). CD spectroscopy is a label-free method suitable for monitoring secondary structure and conformational changes in proteins. DSC is a thermal analysis technique for studying how the heat capacity of proteins changes during thermal unfolding. TSA is a high-throughput method for measuring the thermal stability of protein tertiary structure using fluorescent protein-binding probes that detect protein aggregation. Although these techniques monitor different effects accompanying protein unfolding, the T value calculated as a reference wild-type FGF2 versus the FGF2 peptide according to the present invention... m The relative value of the difference is comparable to a change of less than 0.5℃.

[0057] The information presented in this paper is the first public demonstration that certain changes in wild-type FGF2 result in FGF2 mutants exhibiting greater thermal stability and a longer half-life in human cell cultures than the wild-type protein.

[0058] The FGF2 protein for insertion of the substitution described herein according to the invention can be derived from any mammalian source, such as mice, rats, rabbits, primates, pigs, dogs, cattle, horses, and humans, provided that they meet the criteria specified herein, i.e., that they become thermostable while retaining the desired biological activity of wild-type FGF2. Preferably, the FGF2 protein described herein is derived from a human source. However, any biologically active variant of mammalian FGF2 having at least 85%, and most preferably about 96%, 97%, 98%, 99%, or higher, amino acid sequence identity with the human FGF2 protein of SEQ ID NO:2 used as the basis for comparison can be used in the invention.

[0059] According to certain embodiments, the stable FGF2 polypeptide described herein may also contain any other non-FGF peptide sequence or tag known in the art, which may be used to facilitate its detection, purification, labeling to specific tissues or cells, improved solubility, sustained activity, enhanced expression, etc.

[0060] This disclosure also provides characterization of engineered FGF2, evidence of its role in place of the protein, methods for using the protein in human PSC cultures, and culture media containing at least one thermostable FGF2 protein described herein suitable for culturing undifferentiated human PSCs. The human embryonic stem cells (ESCs) used in the embodiments provided herein are derived from blastocyst-stage embryos obtained with informed consent from the donor. Well-characterized human ESC lines (Adewumi et al. 2007, Nat Biotechnol 25, 803-816), passages 29-41, CCTL14 (Centre of Cell Therapy Line) were used. As for human induced pluripotent stem cells (iPSCs), the AM13 line derived from skin fibroblast reprogramming was used at passages 34-41 via transfection with Yamanaka's cocktail and Sendai virus (Krutá et al. 2014, Stem Cells and Development 23, 2443-2454).

[0061] Generally, the techniques and procedures described herein are performed according to the conventional methods provided throughout this document. The terminology and laboratory procedures used herein in molecular biology, biochemistry, analytical chemistry, and cell culture are generally well-known and commonly used in the art.

[0062] Other features, objects and advantages of the present invention will be apparent from the description and claims. Brief description of the attached diagram

[0064] The invention will be better understood when the following detailed description is considered, and aspects and advantages other than those described above will become apparent. Such detailed description refers to the following drawings, in which:

[0065] Figure 1 It is a polypeptide of wild-type FGF2 (SEQ ID No. 2).

[0066] Figure 2 The nucleotide sequence is the wild-type Fgf2 nucleotide sequence located near the upstream sequence of the pET28b vector. The start codon is gray, the His-tag is underlined, the thrombin cleavage recognition site is black, and the restriction sites NdeI and XhoI used for cloning into the pET28b expression vector are underlined. The wild-type Fgf2 coding sequence begins with ATG and ends with TAG.

[0067] Figure 3The expression and purification of single-site FGF2 mutants (R31W, R31L, V52T, H59F, C78Y, N80G, L92Y, C96Y, S109E, R118W, T121K, V125L) are shown on SDS-PAGE gels. Protein markers: 116, 66.2, 45, 35, 25, 18.4, 14.4 kDa. Recombinant FGF2 mutants with a 6x His tag and thrombin cleavage site have a Mw of approximately 19.1 kDa.

[0068] Figure 4 The comparison of the thermal stability of individual single-point FGF2 mutants (R31W, R31L, V52T, H59F, C78Y, N80G, L92Y, C96Y, S109E, R118W, T121K, V125L) measured by differential scanning calorimetry (DSC) is shown. Mutations selected for constructing combinations are highlighted in gray.

[0069] Figure 5 This is an SDS-PAGE of purified FGF2 CS1 and CS2 mutants. Lane 1: Protein markers (116, 66.2, 45, 35, 25, 18.4, 14.4 kDa); Lane 2: Purified FGF2 CS1 with a 6x His tag and thrombin cleavage site (19.1 kDa); and Lane 3: Purified FGF2 CS2 with a 6x His tag and thrombin cleavage site (19.1 kDa).

[0070] Figure 6 The thermal stability of wild-type FGF2 is compared with that of FGF2 CS1 and FGF2 CS2 mutants. Melting temperatures (T0) were determined using DSC. m ).

[0071] Figure 7 This study demonstrates the ability of wild-type FGF2, FGF2 CS1, and FGF2 CS2 to inhibit RCS cell proliferation after incubation at 36.5°C and 41.5°C for two days. RCS cells were seeded in 96-well plates. Data represent the mean of 6 wells versus indicated standard deviation.

[0072] Figure 8This study demonstrates that FGF2 CS2 maintains the undifferentiated morphology of human PSCs. Both human PSCs, namely ESC (CCTL14) and iPSC (AM13), proliferate either as colonies with a feeder layer (A) or as monolayers on Matrigel (B). Although discontinuation of exogenous FGF2 caused significant growth retardation, both wild-type FGF2 and FGF2 CS2 were able to produce undifferentiated colonies (A) and monolayers (B). Scale bar, 100 μm.

[0073] Figure 9 This study demonstrates that FGF2 CS2 maintains the expression of pluripotency markers in human PSCs. Human PSCs, namely ESC (CCTL14) and iPSC (AM13), proliferated either as colonies with a feeder layer (A) or as monolayers on Matter gel (B). Immunostaining was performed against the cellular pluripotency markers Oct4 and Nanog after five passages in each test condition. Negative controls were incubated without primary antibodies. Wild-type FGF2 and FGF2 CS2 similarly supported the expression of Oct4 and Nanog. Scale bar, 100 μm.

[0074] Figure 10 To demonstrate that FGF2 CS2 supports the proliferation of human ESCs. (A) Human ESCs (CCTL14) were proliferated in FGF2 for each test, and cell counts were performed for 4 consecutive days. Representative results from both experiments are shown. Each data point is shown as the mean ± SEM of three wells. (B, C) Unfed monolayers of human ESCs (CCTL14) were adapted to FGF2 for each test and passaged five times. Cell counts were then performed three days after plating and plotted as relative cell counts (B; n=2). Optionally, cells were counterstained with crystal violet six days after plating, and the results were plotted as relative optical density (C; n=3). Columns show the mean, and error bars show the SEM. Student's t-test, ***p<0.001, **p<0.01, *p<0.05.

[0075] Figure 11This study demonstrated the ability of FGF2 CS2 to maintain its biological activity during prolonged incubation at 37°C. Mouse embryonic fibroblast conditioned medium (CM) prepared without exogenous FGF2 was supplemented with 10 ng / mL FGF2 and incubated at 37°C for 1 h, 3 h, 6 h, 12 h, 24 h, 2 d, 3 d, 4 d, or 5 d. FGF2-starved human ESCs (CCTL14) were then treated with CM containing heat-pre-incubated FGF2 for two h, and immunoblotting was performed against phosphorylated ERK1 / 2. Total ERK1 / 2 levels were used as a loading control. While the biological activity of wild-type FGF2 decreased with increasing heat pre-incubation time, heat-stabilized FGF2 CS2 retained full biological activity even after 5 days at 37°C. Representative results from four different experiments are shown.

[0076] Figure 12 This study demonstrated that FGF2 CS2 can maintain pluripotent human ESCs without requiring daily culture medium changes. Human ESC (CCTL14) colonies were grown for 5 generations in the presence of heat-stabilized FGF2 CS2 at standard (4 ng / mL) or reduced (1 ng / mL) FGF2 concentrations. The culture medium was changed only when colonies isolated, i.e., every 3–4 days. Even at reduced FGF2 concentrations, human ESC colonies retained normal morphology (A) and expression of pluripotency markers (Oct4, B).

[0077] Figure 13 This study demonstrates that repeated replenishment of conditioned medium (CM) is not required for FGF2 CS2. To test the long-term stability of FGF2, CM was prepared without additional replenishment after conditioned with feeder cells. Human PSCs (ESCs, CCTL14 and AM13) without feeder cells were proliferated for 5 generations with FGF2 for each test, and the expression of pluripotency markers (A) and proliferation were monitored (B). Oct4 expression remained high in both FGF2 strains (A). Scale bar, 100 μm. FGF2 CS2 showed superior proliferative support compared to wild-type FGF2 (B). Columns show the mean, and error bars show SEM. Student's t-test, ***p<0.001, **p<0.01, *p<0.05.

[0078] Figure 14The preparation of conditioned medium (CM) is shown. To prepare standard CM, complete medium of human PSCs was conditioned continuously for 5–7 days using mitotically inactivated mouse embryonic fibroblasts (mEF), followed by supplementation with 10 ng / mL FGF2 to restore the growth factor concentration due to its degradation (CM I). For most experiments, CM was prepared from FGF2-deficient human PSC medium, and only the final product was supplemented with the required 10 ng / mL FGF2 (CM II). Optionally, to test the long-term thermostability of FGF2, CM was prepared from medium containing 10 ng / mL FGF2, and then no further supplementation was made (CM III).

[0079] Figure 15 This is an example of output data from screening the bioactivity of a mutated FGF2 peptide in crude extract (CE) derived from the FGF2-S152X library. The codes on the X-axis correspond to the wells of the initial microtiter plate. Freshly melted CE pre-incubated at 41.5°C, or FGF2 from CE, was added to rat chondrocytes grown in parallel microtiter plates, to a final volume of 20 ng / mL. -1 The final concentration was determined, and growth inhibition of chondrocytes was compared with that of samples containing controls by measuring the optical density of the cells. Controls: NEG, negative control (empty plasmid); R31L, positive control (plasmid with a single-point mutant exhibiting improved thermal stability); WT, background control (plasmid with wild-type FGF2). Samples from the original clone H5 that showed statistically more significant growth arrest than the background control were selected as positive hits.

[0080] Figure 16 It is through MagneHis TM SDS-PAGE of FGF2 mutant samples identified in a saturated mutagenesis library after purification using the purification system. M, protein markers (116, 66.2, 45, 35, 25, 18.4, 14.4 kDa). The approximately 19.1 kDa band of the recombinant FGF2 mutant with a 6x His tag and thrombin cleavage site is marked in a box.

[0081] Figure 17 This is an SDS-PAGE of purified FGF2 CS3, CS4, and CS5 mutants. Protein markers: 116, 66.2, 45, 35, 25, 18.4, 14.4 kDa. The recombinant FGF2 mutant with a 6x His tag and thrombin cleavage site has a Mw of approximately 19.1 kDa.

[0082] Figure 18 Proliferation of NIH / 3T3 cells induced by FGF2 CS4 recombinant protein. Example

[0083] The following examples illustrate embodiments of the invention. The examples given are illustrative in nature and not intended to be limiting. While similar or equivalent methods and materials to those described herein can be used in the testing of the invention, suitable methods and materials are described below.

[0084] Example 1. Predicting the stabilizing effect of single-point mutations in FGF2 using an energy-based method.

[0085] resolution higher than Available structures of FGF2 were downloaded from the RCSB protein database (Berman et al., (2000). Nucleic Acids Res. 28, 235–242.). Structures were prepared for analysis by removing ligands and water molecules. In the case of multi-chain structures, one chain was selected. All structures were renumbered so that they started from position 1. Protein side chains were minimized and scored to determine if the minimization was correctly transmitted. The stability effect of all possible single-point mutations was estimated using force field calculations. The ΔΔG free energy was collected and averaged for all structures used, and then averaged for all 20 mutations at specific positions. Evolutionary conservation was estimated using phylogenetic analysis of homologous sequences. Mutations with ΔΔG < -1.0 kcal / mol and conservation < 8 were selected for further analysis. Optimal positions with limited effects on functional regions (such as heparin-binding residues) were identified. Nine single-point substitutions were selected for experimental construction and characterization: R31W, R31L, H59F, C78Y, L92Y, C96Y, R118W, T121K, and V125L (Table 1). These mutants are numbered to correspond to the wild-type human FGF2 sequence (SEQ ID NO:2 below).

[0086] Table 1. Stabilizing mutations selected based on free energy prediction, conservation analysis, and visual inspection.

[0087] residues Location mutation ΔΔG[kcal / mol] Conservatism Functional effects R 31 L -3.6 7 - R 31 W -4.0 7 - H 59 F -2.6 3 FGF-2 dimerization C 78 Y -1.5 3 - L 92 Y -2.3 7 - C 96 Y -3.0 3 Self-association R 118 W -1.6 3 - T 121 K -1.5 7 - V 125 L -1.7 7 -

[0088] ΔΔG: Change in Gibbs free energy after mutation

[0089] Example 2. Predicting the stabilizing effect of single-point mutations in FGF2 using an evolution-based approach.

[0090] Multiple sequence alignments of FGF2 with related proteins were constructed. FGF2 protein sequences were used as the query for PSI-BLAST (Altschul et al., (1997). Nucleic Acids Res. 25, 3389–3402) retrieval against the NCBI nr database. Sequences collected after three iterations were clustered using CD-HIT (Li & Godzik, (2006). Bioinformatics 22, 1658–1659) with a 90% identity threshold. Using default parameters and different p-value thresholds, the resulting dataset of over 500 sequences was clustered using CLANS (Frickey & Lupas, (2004). Bioinformatics. 20, 3702–3704). Sequences with FGF2 at a p-value threshold of 10 were selected. -30 Sequences clustered by p-value were aligned using the MUSCLE program (Edgar, (2004). BMC Bioinformatics. 5, 113.). The final alignment of 238 sequences served as input for a back-to-consensus sequence analysis using a simple consensus approach. This analysis was performed with a consensus cutoff of 0.5, meaning that a given residue must be present at a given position in at least 50% of all analyzed sequences to be designated as a consensus residue. The stabilizing effect of all possible single-point mutations in the FGF2 protein was estimated by free energy calculation. Mutations with a predicted mean ΔΔG ≤ 1 kcal / mol obtained by both methods were considered hotspots for FGF2 stabilization. Functionally important sites of FGF2 were excluded as potentially harmful mutations for biological function. Table 2 summarizes the results of the back-to-consensus analysis. The numbers correspond to the sequence of wild-type human FGF2 (SEQ ID NO: 2 below). Ten mutations were excluded based on the high predicted ΔΔG values, and three mutations were discarded from the design due to their location at functionally important sites for heparin binding. Finally, three single-point mutations passed all criteria and were selected for experimental construction and characterization: V52T, N80G, and S109E.

[0091] Table 2: Mutations that returned to the common sequence identified in FGF2 using the 50% common sequence cutoff. Mutations selected for experimental construction are highlighted in gray.

[0092]

[0093]

[0094] Freq: Frequency of a given FGF-2 residue at a given position in multiple sequence alignment; RES_Top: The most conserved residue at a given position in multiple sequence alignment; Freq_TOP: Frequency of the most conserved residue at a given position in multiple sequence alignment; ΔΔG: Change in Gibbs free energy after mutation.

[0095] Example 3: Construction of twelve single-point mutants of FGF2 and their homogeneity by affinity chromatography

[0096] Commercially synthesized mutants FGF2R31W, R31L, V52T, H59F, C78Y, N80G, L92Y, C96Y, S109E, R118W, T121K, and V125L were subcloned into the NdeI and XhoI sites of pET28b-His-thrombin downstream of the inducible T7 promoter. The resulting constructs were transformed into *Escherichia coli* Dh5α competent cells. Cells were plated in a solution containing kanamycin (50 μg / mL). -1 The cells were plated on agar plates containing kanamycin and grown overnight at 37°C. The plasmid was isolated and its nucleotide sequence was confirmed by commercial sequencing. *E. coli* BL21(DE3) cells were transformed with the expression vector, plated on agar plates containing kanamycin, and grown overnight at 37°C. Single colonies were inoculated into 10 mL of agar containing kanamycin (50 μg / mL). -1 Cells were cultured in LB medium containing kanamycin and grown overnight at 37°C. The overnight culture was then inoculated into 200 mL of LB medium containing kanamycin. Cells were cultured at 37°C. Expression was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.25 mM. Cells were then cultured overnight at 20°C. At the end of the culture, the biomass was harvested by centrifugation and washed with buffer (20 mM dipotassium hydrogen phosphate and potassium dihydrogen phosphate, pH 7.5, 0.5 M NaCl, 10 mM imidazole). Cells in the suspension were disrupted by sonication, and the cell lysates were centrifuged. Proteins were purified from the crude extract using single-step nickel affinity chromatography. The presence of proteins in the peak fraction was confirmed by SDS-PAGE on a 15% polyacrylamide gel. Protein precipitation was minimized by dialyzing in a buffer containing 500–750 mM NaCl. Affinity chromatography was used to purify the FGF2 mutant to produce homogeneous protein with a purity higher than 90%, as determined by SDS-PAGE analysis. Figure 3 The yield of purified FGF2 mutants ranged from 15 to 90 mg / L. -1 .

[0097] Example 4: Determination of the thermal stability of a single-point FGF2 mutant by differential scanning calorimetry

[0098] The thermal stability of single-point FGF2, predicted by energy-based and evolution-based methods, was determined by differential scanning calorimetry (DSC). Thermal desorption stacks of 1.0 mg / mL protein solutions in 50 mM phosphate buffer (pH 7.5) were performed using 500–750 mM sodium chloride, followed by monitoring of the thermal capacity using a VP-capillary DSC system. Measurements were taken at a heating rate of 1 °C / min at temperatures ranging from 20 to 80 °C. m The temperature at which the heat capacity curve reaches its maximum value was determined. The results are shown in Table 4 and... Figure 4 middle.

[0099] Table 4: Thermal stability of FGF2 mutants as determined by differential scanning calorimetry. Mutations selected for constructing the 3- and 6-point mutants are highlighted in gray (see Example 5).

[0100]

[0101] T m Melting temperature; ΔT m Changes in melting temperature after the mutation; 1 The average values ​​from three independent experiments are presented (standard deviation less than 10%).

[0102] This embodiment demonstrates that the computer prediction method of this disclosure can be used to predict stabilization mutations in FGF2. Free energy calculations were performed on the 3-point (R31L, V52T, and H59F) and 6-point (R31L, V52T, H59F, L92Y, C96Y, and S109E) mutants FGF CS1 and FGF2 CS2, respectively, and T... m Combine mutations that increase temperature by at least 2°C (see Example 5).

[0103] Example 5: Construction, purification and thermostability analysis of 3-point FGF2 CS1 and 6-point FGF2 CS2 mutants

[0104] A commercially synthesized multipoint mutant of FGF2 was subcloned into the NdeI and XhoI sites of pET28b-His-thrombin downstream of the inducible T7 promoter (mutated nucleotide and polypeptide sequences are shown in SEQ ID NO:29 to SEQ ID NO:32 below). The resulting construct was transformed into *E. coli* Dh5α competent cells. Cells were plated in a solution containing kanamycin (50 μg / mL). -1The cells were plated on agar plates containing kanamycin and grown overnight at 37°C. The plasmid was isolated and its nucleotide sequence was confirmed by commercial sequencing. *E. coli* BL21(DE3) cells were transformed with the expression vector, plated on agar plates containing kanamycin, and grown overnight at 37°C. Single colonies were inoculated into 10 mL of agar containing kanamycin (50 μg / mL). -1 Cells were cultured in LB medium containing kanamycin and grown overnight at 37°C. The overnight culture was then inoculated into 200 mL of LB medium containing kanamycin. Cells were cultured at 37°C. Expression was induced to a final concentration of 0.25 mM with IPTG. Cells were then cultured overnight at 20°C. At the end of the culture, the biomass was harvested by centrifugation and washed with buffer (20 mM dipotassium hydrogen phosphate and potassium dihydrogen phosphate, pH 7.5, 0.5 M NaCl, 10 mM imidazole). Cells in the suspension were disrupted by sonication, and the cell lysates were centrifuged. Proteins were purified from the crude extract using single-step nickel affinity chromatography. The presence of protein in the peak fraction was confirmed by SDS-PAGE on a 15% polyacrylamide gel. Figure 5 Protein precipitation was minimized by dialyzing in a buffer containing 750 mM NaCl. Yields for both mutants were approximately 20 mg / L of culture. DSC was used to characterize protein thermostability. For DSC experiments, the FGF2 mutant was diluted to 1.0 mg / mL. -1 DSC data collection was conducted within a temperature range of 20℃–100℃. After manually setting the baseline, T... m The results were evaluated as being at the top of the Gaussian curve. The FGF2 CS1 and CS2 mutants exhibited T values ​​of 62.8℃ and 68.0℃, respectively. m ( Figure 6 ).

[0105] Example 6: Thermostability determination of 3- and 6-point FGF2 mutants using rat chondrosarcoma growth arrest assay

[0106] Rat chondrosarcoma (RCS) cells are an immortalized, phenotypic, and stable cell line that responds to small concentrations of FGF with effective growth arrest accompanied by significant morphological changes and extracellular matrix degradation. The FGF receptor (FGFR) acts as a cell proliferation inhibitor in this cell line. To inhibit cell proliferation, FGF mutants must specifically induce FGFR signaling, thus allowing for the measurement of FGF activity as a concentration-dependent reflection of induced growth arrest. A major advantage of RCS assays is the exclusion of toxic chemicals and false positives. High-throughput growth arrest assays were performed in 96-well plates, and cell counts were determined by simple crystal violet staining. Medium containing approximately 40 ng / mL of mutant FGF2 or non-mutant FGF2 was incubated at 36.5°C and 41.5°C for 48 h, mixed every 12 h during this period. To evaluate FGF2 mutant degradation, pre-incubated medium was mixed with mutant FGF2 as a fresh control. One day prior to treatment, RCS cells were seeded in 96-well plates at a concentration of 250 cells per well. Cells were treated for 4 days with a final concentration of 20 ng / mL of pre-incubated FGF2 and fresh controls of each FGF2 mutant. Cells were washed with PBS, fixed with 4% paraformaldehyde, washed again, and stained with 0.025% crystal violet for 1 h. Stained cells were washed three times with distilled water. The stain from the cells was dissolved in 33% acetic acid. Absorbance was measured at 570 nm. Results of the RCS growth stagnation assay are shown below. Figure 7 This example demonstrates that the ability of the 6-point FGF2 CS2 mutant to inhibit RCS cell proliferation remains unaffected even after incubation at 41.5°C for 2 days. In contrast, the bioactivity of wild-type FGF2 was reduced after incubation at 36.5°C.

[0107] Example 7: Thermally stabilized 6-point FGF2 CS2 supports the undifferentiated growth of human pluripotent stem cells.

[0108] To evaluate the ability of the heat-stabilized FGF2 CS2 mutant to support the long-term proliferation of undifferentiated human pluripotent stem cells (PSCs), two culture systems were used: (i) colony growth in the presence of mouse embryonic fibroblast (mEF) feeder layers, and (ii) Matrigel. TM hESC-limited monolayer growth on Matric (BD Biosciences) without feeder cells. Under feeder cell-dependent conditions, the medium consisted of DMEM / F12 (1:1) supplemented with 15% knockout serum substitute, 1% MEM non-essential amino acids, 0.5% penicillin-streptomycin, 100 μM β-mercaptoethanol, and 4 ng / mL wild-type FGF2 or FGF2 CS2 mutant. In the feeder cell-free monolayer system, human PSC growth requires mEF-conditioned medium. For this purpose, the medium was supplemented only with the tested FGF2 (10 ng / mL) (CM II, ...) after feeder cell conditioned. Figure 14Human PSCs were grown for 5 generations under each test condition, and cell morphology and the expression of pluripotency markers Oct4 and Nanog were monitored. Human PSCs maintained in FGF2-free medium produced small differentiated colonies, indicating the important role of FGF2 in maintaining the undifferentiated state of human PSCs. When grown in the presence of FGF2 in both tests, human PSCs exhibited typical morphology—tightly packed colonies as seen when grown with feeder cells, and a high nucleus-to-cytoplasm ratio in both culture systems. Figure 8 No differences in cell morphology were observed between wild-type FGF2 and the 6-point FGF2 mutant. To examine the pluripotency status of human PSCs in more detail, the expression of the pluripotency markers Oct4 and Nanog was tested by immunocytochemistry. No differences in the expression levels or patterns of Oct4 or Nanog were observed under any testing conditions. Figure 9 ).

[0109] Example 8: Thermally stabilized 6-point FGF2 CS2 stimulation of human pluripotent stem cell proliferation

[0110] To determine the proliferation rate, two methods were used. First, the number of human ESCs without feeder cells was counted for four consecutive days after plating. FGF2 in both tests supported the growth of human ESCs with similar efficiency. Figure 10 A). To test the long-term support capacity of FGF2, human ESCs without feeder cells were passaged five times for FGF2 in each test. Then, direct cell counting was used ( Figure 10 B) or the optical density of cells counterstained with crystal violet ( Figure 10 C) to measure proliferation. In these assays, the 6-point FGF2 CS2 mutant supported human ESC proliferation better than wild-type FGF2. These data suggest that thermally stabilized FGF2 CS2 has a significant pro-proliferative effect during both short-term and long-term proliferation.

[0111] Example 9: Thermally stabilized 6-point FGF2 CS2 maintains its bioactivity during long-term incubation at 37°C.

[0112] FGF receptors and their downstream effectors (including ERK1 / 2) are activated upon treatment with FGF2, thereby contributing to the pluripotency of human PSCs (Dvorak et al. 2005, Stem Cell 25, 1200-1211; Eiselleova et al. 2009, Stem Cell 27, 1847-1857). When the bioactivity of FGF2 decreases at 37°C, ERK1 / 2 phosphorylation decreases and human PSCs readily initiate differentiation. To test the thermostability of wild-type FGF2 and FGF2 CS2 mutants, CM prepared without FGF2 was supplemented with 10 ng / mL of the required FGF2 and incubated at 37°C for 1 h, 3 h, 6 h, 12 h, 24 h, 2 d, 3 d, 4 d, or 5 d. Then, FGF2-starved human ESCs were treated with CM containing heat-pre-incubated FGF2 for two h, and Western blotting was performed targeting phosphorylated ERK1 / 2. Although the bioactivity of wild-type FGF2 decreased with heat pre-incubation time, the heat-stabilized FGF2CS2 mutant retained full bioactivity even after 5 days at 37°C. Figure 11 ).

[0113] Example 10: Daily replacement of the culture medium for non-thermally stabilized FGF2 CS2

[0114] Due to the instability of wild-type FGF2, daily culture medium replacement is necessary to maintain the pluripotency of human PSCs. Therefore, we tested whether using a heat-stabilized FGF2 CS2 mutant would circumvent this requirement. To this end, human ESCs were plated on feeder cells in medium containing either standard 4 ng / mL or reduced 1 ng / mL FGF2 mutant, and colonies were allowed to grow for the following 3–4 days without changing the medium. Figure 12 The results show that even at a concentration of 1 ng / mL, the thermally stabilized FGF2 CS2 mutant maintains the undifferentiated morphology of human ESCs and the expression of the pluripotency marker Oct4, without requiring daily culture medium changes.

[0115] Example 11: Repeated replenishment of conditioned medium is not required for the heat-stabilized FGF2 CS2.

[0116] Because wild-type FGF2 is inactivated and degraded during CM preparation, the culture medium needs to be supplemented with FGF2 before and after conditioning with feeder cells. Therefore, we tested the ability of thermostable 6-point FGF2 mutants to maintain undifferentiated growth of human PSCs (CM III, without additional culture medium supplementation after conditioning with feeder cells). Figure 14Human PSCs without feeder cells were proliferated for 5 generations in both wild-type and FGF2 mutant cases, and the expression and proliferation of pluripotency markers were monitored. Although the expression of pluripotency markers was not affected (…),… Figure 13 A), but compared with wild-type FGF2, the 6-point FGF2 mutant showed excellent ability to support proliferation ( Figure 13 B).

[0117] Example 12: Prediction and construction of stable FGF2 mutants by saturation mutagenesis

[0118] Force field calculations were used to identify additional stabilization mutations that could be used for saturation mutagenesis. Mutations were categorized into three groups based on predicted changes in Gibbs free energy (ΔΔG). Mutations with ΔΔG < -1.0 kcal / mol were classified as stable, those with 1.0 < ΔΔG < -1.0 were neutral, and those with ΔΔG > 1.0 were unstable. Eleven sites (K30, E54, E67, C78, ​​R90, S94, C96, E108, N113, T121, and S152) with the highest number of stabilization mutations and the lowest number of destabilization mutations were selected for saturation mutagenesis (Table 5).

[0119] Table 5: Stabilization and destabilization mutations at selected sites in FGF2 predicted by an energy-based method.

[0120]

[0121] All 11 site-saturated mutagenesis libraries of FGF2 were prepared by gene synthesis. Wild-type Fgf2 cDNA fused into the pET28b vector contained a 6xHis tag and a thrombin cleavage recognition site at its N-terminus. Figure 2 These libraries were used as templates for mutagenesis. They were constructed using a "fixed oligomer" technique, which allows only 20 protein amino acids to appear at positions corresponding to degenerate codons in the nucleotide sequence. The libraries were delivered as lyophilized plasmid DNA. The DNA clumps were dissolved in sterile water to 250 ng / μL. -1 The final concentration was determined. 1 μl volumes from each library were electroporated into 100 μl of freshly prepared *E. coli* XJb(DE3) autolysed cells. The cells were then plated onto a substrate containing a final concentration of 50 μg / mL. -1 Eleven separate LB agar plates containing kanamycin were incubated overnight at 37°C. A single colony from each of the eleven LB agar plates was used to inoculate 250 μl of kanamycin (50 μg / mL). -11 mL of LB medium was added to individual wells of a 96-well plate. The plate was incubated overnight at 37°C with shaking at 200 rpm in a high humidity chamber. The culture was then added to each well to a final concentration of 50 μg / mL. -1 Expression was induced in fresh LB medium containing 0.25 mM and 3 mM kanamycin, IPTG, and L-arabinose. The plates were incubated overnight at 20°C with shaking. After 22 hours, the plates were centrifuged and the supernatant was discarded. The entire microtiter plate containing cell clumps was frozen at -70°C. Then, 100 μl of lysis buffer (20 mM sodium phosphate buffer, 150 mM NaCl, pH 7.0) was added to each well, and the plate was incubated at 30°C for 20 min. Cell debris was removed from the resulting cell lysates, and the total soluble protein content of each plate was determined using the Bradford method. The FGF2 content, expressed as a percentage of total soluble protein, was determined by SDS-PAGE and densitometric assay. The concentration of total soluble protein in the selected crude extract samples from each library ranged from 0.2 to 0.3 mg / mL. -1 The FGF2 content in the crude extract ranged from 5% to 7% of the total soluble protein.

[0122] The bioactivity of cell lysates containing various FGF2 mutants was determined using RSC via growth arrest assay. Microtiter plates containing crude extracts of FGF2 mutants and controls were thawed at room temperature and pre-incubated at 41.5°C for 48 hours. The pre-incubated crude extracts were added to chondrocytes grown in fresh microtiter plates to a final concentration of 20 ng / mL. -1 Furthermore, the inhibition of chondrocyte growth was compared with that of a control sample by measuring the optical density of the cells. Figure 15 The more stable the FGF2 mutant in the added crude extract, the more pronounced the growth inhibition. Growth inhibition in samples not pre-incubated at elevated temperatures was also measured. Samples exhibiting more significant growth inhibition than those containing wild-type FGF2 were considered positive hits. For each positive hit, the entire screening procedure described above was repeated. The mutated Fgf2 gene was sequenced using the Sanger method. The resulting sequences were compared with the wild-type FGF2 sequence to verify the inserted mutation (Table 6).

[0123] Table 6. Overview of the screening results of 11 saturated mutagenesis libraries from FGF2.

[0124]

[0125]

[0126] Escherichia coli BL21(DE3) cells were transformed with the expression vector pET28b-His-thrombin::fgf2x (x = 32 different FGF2 mutants) and plated on a substrate containing kanamycin (50 μg / mL). -1 Cells were cultured overnight at 37°C on agar plates. Single colonies were inoculated into 10 mL of LB medium containing kanamycin and grown overnight at 37°C. Expression was induced with IPTG at a final concentration of 0.25 mM. Cells were then cultured overnight at 20°C. At the end of culture, the biomass was centrifuged and cell pellets were frozen at -70°C. The pellets were thawed and resuspended in FastBreak. TM Cell lysis reagent 1X (FastBreak) TM Cell Lysis Reagent 1X). Lysed cells were incubated on a shaking platform at room temperature for 10–20 minutes. MagneHis TM Ni particles were added to the cell clumps. This was to improve compatibility with MagneHis. TM The binding of Ni particles involved adding 500 mM NaCl to a specific volume of bacterial culture (0.03 g NaCl per 1.0 mL of lysate). The tube containing the lysed bacterial cells was incubated at room temperature for 2 minutes, then placed on a magnetic rack for approximately 30 seconds to capture MagneHis. TM Ni particles. Carefully remove the supernatant. To wash away unbound cellular proteins, add MagneHis solution with 500 mM NaCl. TM Binding / washing buffer. Carefully remove the supernatant. Repeat the washing step twice. Elution of binding proteins is achieved by adding 105 μl of MagneHis solution containing 500 mM NaCl. TM Elution buffer (MagneHis) TM Elution Buffer was used. The elution mixture was incubated at room temperature for 2 minutes, and then the tubes were placed on a suitable magnetic rack for about 30 seconds to remove the supernatant containing the purified protein. The presence of all FGF2 mutants was confirmed by SDS-PAGE on a 15% polyacrylamide gel. Figure 16 The yield of purified FGF2 mutants ranged from 10 to 100 mg / L. -1 Most FGF2 mutants are expressed at levels similar to or higher than wild-type FGF2.

[0127] The thermal stability of the target protein was measured using a thermal displacement assay. Measurements were performed in 96-microtiter plates. Each well consisted of 2 μL of Sypro Orange dye (diluted 40-fold in water) and an appropriate volume of FGF2 mutant calculated using the following equation:

[0128] VFGF2var=(CFGF2var*Vdv) / Cdc

[0129] VFGF2var=(CFGF2var*1) / 2.5

[0130] Where VFGF2var is the volume of the FGF2 mutant, CFGF2var is the concentration of the FGF2 mutant, and Cdc is the defined concentration of 2.5 mg / mL. -1 The volume of elution buffer was defined as 1 μL. Elution buffer was then added to bring the total volume in the wells to 25 μL. Thermal denaturation was measured on a real-time PCR system, starting at 25 °C and gradually increasing in 1 °C increments to a final temperature of 95 °C. m The value is generated by the Boltzmann derivation, where T m The values ​​are taken from the inflection point of the fluorescence melting curve (Table 7).

[0131] Table 7: Thermal stability of FGF2 mutants from saturated mutagenesis, determined by thermal shift measurement. T0 of wild-type FGF2, determined by thermal shift measurement. m The temperature was 51°C. The amino acid substitutions selected for further calculations and analyses (see Example 13) are highlighted in gray.

[0132]

[0133] T m Melting temperature; ΔT m : Change in melting temperature after mutation; nd, not measured due to poor protein folding.

[0134] Example 13: Combination of single-point mutants from saturation mutagenesis

[0135] Force field calculations were used to determine combinatorial mutations without antagonistic effects and to design highly stable multi-site mutants of FGF2. The following mutations from library screening (see Example 12) were selected for further analysis: K30I, E54D, S94I, C96N, E108H, and T121P. These mutations were combined with existing mutations from the FGF2 CS2 mutant (R31L, V52T, H59F, L92Y, C96Y, and S109E). All combinations of two-site mutants were computer-generated to predict the additivity of each mutation. The difference between the predicted ΔΔG and the sum of the individual single-site mutations was >1 kcal·mol⁻¹. -1Two-point mutants were considered antagonistic. Therefore, three different multi-point mutants were designed for further characterization. All three mutants were based on the previously designed FGF2 CS2. The FGF2 CS3 mutants (R31L, V52T, H59F, L92Y, C96Y, S109E, K30I, E54D, and E108H) contained three additional mutations that exhibited the highest stabilizing effect in thermal displacement assays. FGF2CS4 (R31L, V52T, H59F, L92Y, S109E, E54D, S94I, C96N, and T121P) were designed to preserve protein function. All mutations targeting the interface between FGF2 and FGFR1 or the FGFR2 receptor, or those at sites important for dimerization, were discarded, and the C96Y mutation was replaced with C96N due to its superior, experimentally confirmed stabilizing effect. FGF2 CS5 mutants (R31L, V52T, H59F, L92Y, S109E, K30I, E54D, S94I, C96N, E108H, and T121P) were selected to maximize the thermal stability effect of the protein, which contained all the mutations found to stabilize FGF2 in thermal displacement assays (Example 12).

[0136] Example 14: Construction, purification and thermal stability analysis of FGF2 CS3, CS4 and CS5 mutants

[0137] A commercially synthesized multipoint mutant of FGF2 was subcloned into the NdeI and XhoI sites of pET28b-His-thrombin downstream of the inducible T7 promoter (mutated nucleotide and polypeptide sequences are shown in SEQ ID NO:33 to SEQ ID NO:38). The resulting construct was transformed into *E. coli* Dh5α competent cells. Cells were plated in a solution containing kanamycin (50 μg / mL). -1 The cells were plated on agar plates containing kanamycin and grown overnight at 37°C. The plasmid was isolated and its nucleotide sequence was confirmed by commercial sequencing. *E. coli* BL21(DE3) cells were transformed with the expression vector, plated on agar plates containing kanamycin, and grown overnight at 37°C. Single colonies were inoculated into 10 mL of agar containing kanamycin (50 μg / mL). -1Cells were cultured in LB medium containing kanamycin and grown overnight at 37°C. The overnight culture was then inoculated into 200 mL of LB medium containing kanamycin. Cells were cultured at 37°C. Expression was induced with IPTG at a final concentration of 0.25 mM. Cells were then cultured overnight at 20°C. At the end of the culture, the biomass was harvested by centrifugation and washed with buffer (20 mM potassium phosphate buffer, pH 7.5, 0.5 M NaCl, 10 mM imidazole). Cells in the suspension were disrupted by sonication, and the cell lysates were centrifuged. Proteins were purified from the crude extract using single-step nickel affinity chromatography. The presence of protein in the peak fraction was confirmed by SDS-PAGE on a 15% polyacrylamide gel. Figure 17 Protein precipitation was minimized by dialyzing in a buffer containing 750 mM NaCl. The mutant yield was between 5 and 10 mg / L. DSC was used to characterize the protein's thermostability. For DSC experiments, the FGF2 mutant was diluted to 1.0 mg / mL. -1 Data collection was performed at a rate of 1 °C / min within a temperature range of 20 °C–90 °C. The FGF2 CS3, FGF2 CS4, and FGF2 CS5 mutants exhibited T values ​​of 72.6, 72.2, and 72.7 °C, respectively. m .

[0138] Example 15: Proliferation of NIH / 3T3 cells using heat-stabilized FGF2 CS4

[0139] NIH / 3T3 cells were cultured in 190 μl of DMEM 31966 medium per well. Seeds were generated at a density of 40,000 cells / cm² in a medium containing +P / S and 10% newborn calf serum. After 24 hours, the medium was replaced with starved medium (DMEM 31966). +P / S +0.5% newborn calf serum). After 16 hours, the cells were diluted in sterile water, treated with FGF2CS4 to a final concentration of 0.01–20 ng / mL, and cultured at 37°C for another 48 hours. Fluorescence assay for cell proliferation ( Figure 18 The experiment was performed in triplicate. As determined in the NIH / 3T3 cell proliferation assay, the EC50 of FGF2 CS4, i.e., the maximum response concentration at which FGF2 CS4 produces half of the target value, is 0.6–1.1 ng / mL.

Claims

1. A thermostable polypeptide having FGF2 activity, said polypeptide consisting of the sequence of any one of SEQ ID NO: 30, 32 and 36.

2. The thermostable polypeptide according to claim 1, wherein said polypeptide has the amino acid substitutions R31L, V52T and H59F.

3. The thermostable polypeptide according to claim 1, wherein said polypeptide has the amino acid substitutions R31L, V52T, H59F, L92Y, C96Y, S109E.

4. The thermostable polypeptide according to claim 1, wherein said polypeptide has the amino acid substitutions R31L, V52T, E54D, H59F, L92Y, S94I, C96N, S109E, T121P.

5. Use of the thermostable polypeptide of any one of claims 1 to 4 for the manufacture of a medicament for regenerative medicine and other medical applications.

6. Use of the thermostable polypeptide of any one of claims 1 to 4 for cosmetics.

7. A culture medium suitable for culturing human pluripotent stem cells in an undifferentiated state, comprising an effective amount of a thermostable polypeptide as defined in any one of claims 1 to 4 in the range of 1.0 ng / µl of culture medium to 100 ng / µl of culture medium.

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