WO2022114817A1 - Fgf11 isoform2 polypeptide and use of same - Google Patents

Abstract

Proposed is a polypeptide of SEQ ID NO: 2, which is a wild-type human FGF11b C-terminal-deficient polypeptide. A polypeptide having at least 90% identity with said SEQ ID NO: 2 is effective for cell proliferative activity, and thus is provided as a pharmaceutical composition and a cosmetic composition.

Description

FGF11 isoform 2 polypeptide and uses thereof

The present disclosure relates to FGF11 isoform 2 (hereinafter, FGF11b) polypeptides and uses thereof.

FGF (Fibroblast Growth Factor) is a factor that plays an important role in regulating cell growth, proliferation, and differentiation. Various types of FGFs are generated to maintain the function of each tissue in the human body, and they perform unique functions in cell differentiation and proliferation. However, as aging progresses, the concentration of FGFs in each tissue, such as the skin, is gradually lowered. Accordingly, cell regeneration and division functions are weakened, so that wrinkles are formed in the skin and elasticity is reduced.

Among the various FGFs, FGF11 (Fibroblast Growth Factor 11) is not well known for its function and regulatory mechanism because it is difficult to obtain a stable protein due to difficult protein expression and purification.

The present disclosure intends to provide a FGF11b polypeptide having cell proliferative activity.

The FGF11b polypeptide having cell proliferative activity according to the embodiments is a polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2.

The FGF11b polypeptide according to the Examples exhibits excellent cell proliferative activity.

1 shows the sequence of the FGF11b polypeptide of SEQ ID NO: 1.

2 shows the sequence of the FGF11b polypeptide of SEQ ID NO: 2.

3 shows SDS-PAGE of the FGF11b polypeptide of SEQ ID NO: 1 (hFGF11b) and FGF11b of SEQ ID NO: 2 (hFGF11b-delC).

4 is a graph showing the results of measuring the cell proliferation activity of the FGF11b polypeptide (hFGF11b) of SEQ ID NO: 1 and FGF11b (hFGF11b-delC) of SEQ ID NO: 2.

5 is a graph showing the results of measuring the cell proliferation activity of the FGF11b polypeptide (hFGF11b) of SEQ ID NO: 1 and FGF11b (hFGF11b-delC) of SEQ ID NO: 2 according to time.

Hereinafter, embodiments will be described in detail so that those of ordinary skill in the art can easily implement it. The embodiment may be implemented in various different forms, and is not limited to the specific embodiment described herein.

Unless the definitions of some terms used in this disclosure are otherwise defined below, 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 belongs.

The techniques and processes described in this disclosure are generally performed according to conventional methods, which are presented throughout this application. In general, the nomenclature and laboratory procedures in molecular biology, biochemistry, analytical chemistry and cell culture used in this disclosure are well known in the art and are the same as commonly used.

variant

The present disclosure provides an FGF11b polypeptide with increased cell proliferative activity by mutagenesis. In the present disclosure, mutations were prepared by mutagenesis after predicting an optimal polypeptide that was not previously known through bioinformation analysis and computer-based protein design.

According to the mechanism of action, 22 types of FGF are divided into paracrine, endocrine, and intracrine. Paracrine (paracrine) and endocrine (endocrine) FGFs are secreted out of the cell and act on peripheral cells or distant cells, but endocrine (intracrine) is known to act inside the cell without being secreted out of the cell. The endogenous FGF includes FGF11, FGF12, FGF13, and FGF14, among which the function and regulatory mechanism for FGF11 are not well known.

The reason is that it is difficult to stably secure FGF11 protein due to difficulties in protein expression and purification. Accordingly, the present inventors established the expression, isolation, and purification of FGF11b, and confirmed that the novel FGF11b has cell proliferation activity through an experiment in which the purified protein is treated in cells.

1 shows the wild-type human FGF11b polypeptide sequence.

In the present disclosure, the term 'wild type' refers to a native FGF11b having the most common amino acid sequence among members of the species. In the present disclosure, wild-type FGF11b is human FGF11b, which is a 166 amino acid long (SEQ ID NO: 1) protein.

Figure 2 shows the C-terminal deleted polypeptide sequence (SEQ ID NO: 2) of wild-type human FGF11b according to an embodiment of the present invention.

The present inventors confirmed that the polypeptide having at least 90% identity over the entire length of the C-terminal deletion polypeptide of SEQ ID NO: 2 with the polypeptide of SEQ ID NO: 2 of wild-type human FGF11b is very effective in cell proliferative activity.

In the present disclosure, the percent sequence identity is calculated by determining the number of positions in which identical amino acid residues exist in both sequences, dividing this by the total number of positions in the segment compared to the reference molecule, and multiplying this by 100. , calculated by calculating the % sequence identity. Sequence alignment methods are well known in the art.

In general, the coding gene for FGF11b is cloned and then expressed in a transformed organism, preferably a microorganism. The host organism expresses a foreign gene to produce FGF11b under expression conditions. In addition, synthetic recombinant FGF11b can be made in eukaryotes such as yeast or human cells.

In some embodiments, the FGF11b polypeptide of SEQ ID NO: 2 according to the present invention described herein easily performs detection, purification, tagging to a specific tissue or cell, improved stability, extended activity, improved expression, etc. It may further comprise a sequence or tag other than any additional FGF peptide known in the art, which can be used to

Pharmaceutical and cosmetic compositions

A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 may be provided as a pharmaceutical and/or cosmetic composition together with a pharmaceutically or cosmetically acceptable carrier.

A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 is a subject in need of promoting angiogenesis, promoting wound healing, promoting chondrogenesis or osteogenesis, or promoting neurogenesis or wrinkle improvement, skin elasticity It can be administered to a subject in need of improvement in skin condition, such as improvement, skin aging prevention, hair loss prevention or hair growth promotion, skin moisture improvement, age spots removal, or acne treatment.

The polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 can be administered in "native" form or, if desired, in the form of a salt, ester, amide, prodrug, derivative, etc., provided that Salts, esters, amides, prodrugs or derivatives can be selected from substances that are pharmacologically suitable, ie, effective for the method(s). Salts, esters, amides, prodrugs and other derivatives of peptides are known to those skilled in the art of synthetic organic chemistry and can be prepared using, for example, standard known procedures.

A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 may be formulated for subcutaneous, parenteral, topical, oral, nasal (or otherwise inhaled), rectal, or topical administration, such as an aerosol, cream, It can be formulated as a serum and a transdermal product in the form of a patch. The composition may be administered in various unit dosage forms depending on the method of administration. Suitable unit dosage forms may include, but are not limited to, powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injections, implantable sustained release formulations, lipid complexes, and the like.

The polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 may be combined with a cosmetically acceptable carrier to form a cosmetic composition with a filler (e.g. hyaluron filler, polymethylmethacrylate (PMMA) ) microspheres and collagen fillers) and the like. The composition may preferably be for topical, subcutaneous, or transdermal administration.

The composition may be an injectable composition.

The composition may further comprise collagen (eg bovine, porcine, or human collagen) hyaluronic acid. The collagen may be synthetic collagen, and the hyaluronic acid may be a chicken meal or a fermentation product of a microorganism.

The composition may further comprise an anesthetic (eg, lidocaine).

The composition may be a skin cream (eg, a face cream).

The composition may be a liquid formulation in the form of a serum or toner.

The composition may be a semi-solid preparation in a gel state.

Pharmaceutically acceptable carriers may be approved by a federal or state regulatory agency or may be approved by the U.S. include those listed in the pharmacopoeia or other generally recognized pharmacopeias for use in animals, more particularly in humans or animals, more particularly humans. "Carrier" means, for example, a diluent, adjuvant, excipient, adjuvant or vehicle with which one or more peptides described herein are administered.

A pharmaceutically acceptable carrier may contain, for example, one or more physiologically acceptable compounds that act to stabilize the composition or increase or decrease absorption of the various variants disclosed in Table 1. Physiologically acceptable compounds may include, for example, carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, protective and absorption enhancers such as lipids , a compound that reduces the clearance or hydrolysis of the peptide, or other excipients, stabilizers and/or pH adjusting buffers.

Other physiologically acceptable compounds, particularly used in the manufacture of tablets, capsules, gel caps, and the like, may include, but are not limited to, binders, diluents/fillers, disintegrants, lubricants, and suspending agents.

To prepare an oral dosage form (eg, tablet) excipients, optional disintegrants, binders and optional lubricants, etc. are added to the various variants disclosed in Table 1 and the resulting composition can be compressed. If desired, the compressed product may be coated using known methods for taste masking or enteric dissolution or sustained release.

Other physiologically acceptable compounds that can be formulated with the polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 include wetting agents, emulsifying agents, dispersing agents or preservatives particularly useful for preventing the growth or action of microorganisms. can do. Excipients may be used in a sterile, contaminant-free state.

A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 can be incorporated into formulations for cosmetic use and applied topically and can be applied topically in skin creams (eg, face creams) or body lotions, wrinkle-removing It may be formulated as a cream or incorporated into a cosmetic, sunscreen, or moisturizer.

Also, the polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 may be incorporated into a formulation optionally further comprising a filler, a moisturizer, a vitamin (eg, vitamin E), and/or a colorant/dye agent. can

Suitable injectable cosmetic formulations may include, but are not limited to, formulations that incorporate a polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 together with one or more filler substances. Exemplary materials usable as injectable cosmetic wrinkle fillers include, but are not limited to, temporary (absorbable) fillers such as collagen (eg, synthetic collagen, bovine collagen, porcine collagen, human collagen, etc.), hyaluronic acid gel, calcium hydride. lyxylapatite (typically implanted in the form of a gel), or poly-L-lactic acid (PLLA), and the like. The peptides may also be incorporated into injectable cosmetic formulations containing permanent (non-absorbable) fillers. Exemplary “permanent” fillers may include, but are not limited to, polymethylmethacrylate beads (PMMA microspheres).

A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 may be incorporated into or administered with a dermal filler, an injectable formulation, etc.: Such an injectable formulation may contain an anesthetic (eg, lidocaine or its analogs) may be further included. The injectable formulation is substantially sterile or sterile and/or conforms to institutional guidelines for subcutaneous injectable fillers.

A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 may be administered to a subject using any route known in the art, which route may be administered, for example, by injection (e.g., , intravenous, intraperitoneal, subcutaneous, intramuscular, or intradermal), inhalation, transdermal application, rectal administration, vaginal administration, or oral administration. Preferred routes of administration include subcutaneous, transdermal, or topical application.

An effective amount of a polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 includes, but is not limited to, administration via topical (ie, non-systemic) administration, such as, but not limited to, peripheral intramuscular, intravascular, and subcutaneous administration. It can be administered by peripheral administration.

Administration of the polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 may be in any convenient manner, for example, by injection, intravenous and arterial stents (including eluting stents), catheter, oral administration, inhalation. , transdermal application, rectal administration, and the like.

A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 may be formulated prior to administration with a pharmaceutically acceptable carrier, eg, as described above. A pharmaceutically acceptable carrier is determined in part by the particular composition to be administered, as well as by the particular method used to administer the composition.

The dose administered in a subject should, in the context of the methods described herein, be sufficient to affect a beneficial therapeutic response (eg, increased subcutaneous adipogenesis) in the subject over time. The dose will be determined by the efficacy of the particular vehicle/delivery method employed, the site of administration, the route of administration, and the condition of the subject, as well as the body weight or surface area of the subject being treated. The size of the dose will also be determined by the presence, sex, and extent of any adverse side effects that accompany administration of a particular peptide in a particular subject.

A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 may be administered systemically (eg, orally, or as an injection) according to standard methods well known to those skilled in the art. The peptides can be administered to the oral cavity in various forms, such as lozenges, aerosol sprays, mouthwashes, coated swabs, and the like. A variety of buccal, and sublingual formulations are also contemplated. A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 may be administered as a depot formulation when formulated as an injectable to provide treatment over a period of time.

The polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 may be administered topically, for example, to the skin surface, to a local lesion or wound, to a surgical site, or the like.

A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 can be delivered through the skin using a conventional transdermal drug delivery system, i.e., a transdermal "patch", wherein the entire length of the sequence of SEQ ID NO: 2 is present. Polypeptides having at least 90% identity over their length may be contained within a layered structure that typically serves as a drug delivery device to be attached to the skin.

Other formulations for topical delivery include, but are not limited to, ointments, gels, sprays, fluids, and creams. Ointments may be semi-solid preparations, typically based on petrolatum or other petroleum derivatives. As with any other carrier or vehicle, the ointment base must be inert, stable, non-irritating and non-sensitizing. Creams containing a polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO:2 will typically be a viscous liquid or semi-solid emulsion, often oil-in-water or water-in-oil. Cream bases are typically water washable and contain an oil phase, an emulsifier and an aqueous phase. The particular ointment or cream base to be used is one that will provide for optimal drug delivery, as will be appreciated by those skilled in the art.

The polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 is stored in a storage container prepared for dilution (eg, in a pre-measured volume), or in a large amount of water, alcohol, hydrogen peroxide, or other diluent. may be provided as a "concentrate" in soluble capsules prepared for the addition of For example, the peptide can be lyophilized for later reconstitution.

A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 may have a variety of uses. Polypeptides having at least 90% identity over the entire length of the sequence of SEQ ID NO:2 may find use in many applications. For example, since subcutaneous fat provides fullness and firmness to the skin, enhancing the formation of subcutaneous fat has use in cosmetic surgery procedures. Aging skin contains less subcutaneous fat. Therefore, administering a polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 to a desired site to promote subcutaneous fat formation can result in fuller and younger looking skin. This approach can replace current methods of transplanting fat cells from other parts of the body (eg, thighs or buttocks), a process often with low success rates.

A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 can be used to selectively enhance subcutaneous adipose tissue (eg, subcutaneous fat without substantially increasing visceral fat and/or other adipose tissue). to improve tissue). In response to administration of a polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO:2, adipocyte formation occurs in dermal fibroblasts, and volume can be added within a selected subcutaneous site in the subject.

A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 may be used to reduce scarring. This can be achieved by administering a polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 in an amount sufficient to reduce the scarring area or improve the appearance of the scarring area. A scar can be, for example, a scar produced by a burn, a scar produced by surgery, a scar produced by acne, a scar produced by a biopsy, or a scar produced by an injury.

A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 can be used in various cosmetic procedures, for example, to improve the appearance of the skin. This may be accomplished by administering one or more peptides to the site of the subject in an amount sufficient to improve the appearance of the skin. Such administration may include subcutaneous administration to areas such as the lips, eyelids, cheeks, forehead, chin, neck, and the like. These peptides reduce wrinkles, reduce sagging skin, improve the surface texture of the skin, reduce, remove, fill in wrinkles, remove or reduce age spots, and/or remove dark circles under the eyes etc. can be used. These cosmetic applications are exemplary and not intended to be limiting.

A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO:2 may be used to improve tissue volume at a site of interest. This may be accomplished by administering one or more of the peptides described herein in an amount sufficient to increase tissue volume at the site of the subject. For example, increasing tissue volume may include firming or augmenting breast tissue and/or firming or augmenting hip tissue or other parts of the body or face.

The polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 used in this case may be used in an amount of 0.01 to 10 ppm. Above 10 ppm, there is a possibility of side effects that may induce adverse reactions in excessive amounts. Accordingly, the practical use range is 0.01 to 10 ppm, preferably 0.01 to 2 ppm.

Polypeptides having at least 90% identity over the entire length of the sequence of SEQ ID NO:2 may also be used to smooth the skin in the site of a subject. This can be achieved by administering the peptide in an amount sufficient to soften the skin at the desired site. The smoothing may include smoothing skin scarred by acne, smoothing out areas of cellulite, smoothing or reducing stretch marks, and/or smoothing wrinkles.

A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 may be used to recruit stem cells in a subject to the formation of subcutaneous fat. This can be achieved by administering a polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 in an amount sufficient to recruit stem cells to the formation of subcutaneous fat. This has utility, for example, in various reconstructive surgical procedures and the like.

A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 can be used to reconstruct tissue in a subject. Such reconstruction may include, for example, breast reconstruction (eg, after surgery to remove a tumor), or face or limb reconstruction (eg, after an automobile accident or burn). This can be achieved by administering the various variants disclosed in Table 1 in an amount that increases the volume of the tissue either during or after the tissue reconstruction process. A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 may optionally be used in combination with a tissue graft material or other procedure that enhances the healing of skin or injured tissue.

A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 can be used to reduce heel pain in a subject by administering it in an amount sufficient to reduce heel pain experienced by the subject when walking.

A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 may be administered for augmentation of subcutaneous fat to increase thermoregulation and/or improve immune function. The subject is a poly(a) having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 for preventing disease or treating an ongoing disease associated with increased organ fat, including but not limited to cardiovascular disease, and other obesity associated diseases. can be treated with peptides.

Administration in any of these methods may be topical or systemic, and may be by any route described herein, such as topical, subcutaneous, transdermal, oral, nasal, vaginal, and/or rectal administration. Preferably, the polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 can be administered by subcutaneous injection. Alternatively, the polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 may be administered topically in the form of a skin cream such as a face cream, or administered transdermally via a transdermal patch.

Although the above uses and methods are described with reference to use in humans, they are also suitable for use in animals, eg, veterinary use. Accordingly, certain preferred organisms include, but are not limited to, humans, non-human primates, canines, horses, cats, pigs, ungulates, rabbits, and the like.

badge

The polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO: 2 is included in a 'medially effective amount' corresponding to the amount necessary to maintain the pluripotent stem cells in an undifferentiated state for at least 5 passages. and can be provided as a human pluripotent stem cell medium.

In the present disclosure, including both human embryonic stem cells and induced pluripotent stem cells, the term 'human pluripotent stem cells' refers to pluripotent stem cells that are capable of generating virtually all cell types in the human body with the same progeny. It is characterized by the ability to form a self-renewal capacity.

In the present disclosure, the term 'maintaining stem cells in a pluripotent state' means maintaining the cells in an undifferentiated state having the ability to differentiate into virtually any cell type. This pluripotent state relies on a stemness-supporting cocktail of growth factors, the most important growth factor being a polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO:2. Polypeptides having at least 90% identity over the entire length of the sequence of SEQ ID NO:2 support self-renewal in several ways: directly activating the mitogen-activated protein kinase pathway, transforming growth factor β1 and activin It indirectly catalyzes signaling (Greber, et al. 2008, Stem Cells25, 455-464). FGF2, through cell adhesion and survival functions, contributes to the pluripotency of human PSCs complexly (Eisellova, et al. 2009, Stem Cells 27, 1847-1857).

The present disclosure relates to the characterization of a polypeptide having at least 90% identity over the entire length of the sequence of the engineered subject SEQ ID NO: 2, demonstrating the effect of substitution in the protein, methods of using the protein in human PSC culture, and methods of using undifferentiated human PSCs. Provided is a medium comprising one or more polypeptides described herein suitable for culturing in a state. Human embryonic stem cells (ESCs) used in the examples provided herein were derived from blastocyst embryos obtained with the informed consent of the physician. A well-characterized human ESC cell line (Adewumi, et al. 2007, Nat Biotechnol 25, 803-816) CCTL14 (Center of Cell Therapy Line) was used at passages 29-41. As in human induced pluripotent stem cells (iPSCs), the AM13 cell line, derived using Yamanaka's cocktail and reprogramming of dermal fibroblasts by Sendai virus transfection, was used as the 34-41 cell line. Accounting status was used (Kruta et al. 2014, Stem Cells and Development 23, 2443-2454).

Hereinafter, preferred experimental examples are presented to help the understanding of the present invention, but the following experimental examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

Experimental Example 1: Construction and purification of mutants using the pET3a vector

SEQ ID NO: 1 (hFGF11b) and SEQ ID NO: 2 (hFGF11b-delC) were synthesized and subcloned into pET3a vector. The recombinant vector into which FGF11b was inserted was transformed into Rosetta(DE3)pLysS cells and expressed.

10ml LB media (Ambrothia) (0.25g used) was inoculated and 10 μl of ampicillin (Ampicillin, 50mg/ml) was added, and then pre-cultured at 37°C and 180rpm conditions.

1 ml of the pre-culture solution and 100 μl of ampicillin (Ampicillin, 50 mg/ml) were inoculated into 1 L LB media (ambrothia) (25 g used), and the main culture was performed at 37° C. and 180 rpm. In the case of two flasks, 0.5 mM of beta-di-1-thiogalactopyranoside (β-Dl-thiogalactopyranoside; IPTG) was added when the OD ₆₀₀ value was 0.5 at 37°C (180rpm) and 30°C (180rpm). ) was induced for 2 hours 30 minutes and 5 hours, respectively.

And, in the other flask, when the value of OD ₆₀₀ was 0.6, 0.5 mM beta-di-1-thiogalactopyranoside was added to induce expression at 20° C. (130 rpm) for 20 hours.

The expressed hFGF11b and hFGF11b-delC were dissolved in a lysis buffer solution (20 mM Tris pH 8.0, 100 mM Ammonium Sulfate, 1 mM DTT, 5% glycerol), sonicated, and centrifuged at 13,000 r.p.m for 30 minutes, followed by purification.

After centrifugation, the optimally dissolved supernatant was injected into a column with Heparin beads. The hFGF11b and hFGF11b-delC proteins injected into the column were bound to heparin beads and washed with a buffer solution (20 mM Tris pH 8.0, 200 mM Ammonium Sulfate, 1 mM DTT, 5% glycero) three times the volume. As the elution buffer, the first elution buffer solution (20 mM Tris pH 8.0, 100 mM Ammonium Sulfate, 1 mM DTT, 5% glycerol) and the second elution buffer solution (20 mM Tris pH 8.0, 1000 mM Ammonium Sulfate, 1 mM DTT, 5% glycerol) was used to elute the protein by gradient (0.1 to 1M gradient).

Finally, the protein fraction of hFGF11b and hFGF11b-delC was purified by gel filtration using a buffer (20 mM Tris pH 8.0, 100 mM Ammonium Sulfate, 1 mM DTT, 5% glycerol) on a HiLoad™ 16/60 Superdex 75 (Amersham Biosciences) column. did.

Purified hFGF11b and hFGF11b-delC proteins were analyzed with FGF11b (2.93 mg/ml) dissolved in a buffer (20 mM Tris pH 8.0, 100 mM Ammonium Sulfate, 5% glycerol) to confirm stabilization by ammonium sulfate. 1 ml was dialyzed against 1 L of 100 mM ammonium sulfate-free solution (20 mM Tris pH 8.0, 5% glycerol) in a refrigerator at 4° C. for 20 hours.

As exemplified in FIG. 3 , hFGF11b and hFGF11b were added to 0.5 mM beta-di-1-thiogalactopyranoside (β-D-l-thiogalactopyranoside; IPTG) and the expression was induced at 20° C. for 20 hours. - It was confirmed that the delC protein was expressed in a water-soluble state.

In addition, in order to confirm the importance of ammonium sulfate in the purification process, it was confirmed that hFGF11b protein was aggregated when dialyzed in a solution (20 mM Tris pH 8.0, 5% glycerol) in which ammonium sulfate was removed. Through this, it was confirmed that ammonium sulfate increases the stability of hFGF11b.

Experimental Example 2: Confirmation of cell proliferation activity of hFGF11b and hFGF11b-delC

BALB3T3 cells were used, and cultured and maintained in DMEM medium containing 10% bovine serum. In order to confirm the cell proliferation activity by hFGF11b and hFGF11b-delC, the cells were insulin 10 μg/mL, dexamethasone 1 μM, transferrin 10 μg/mL, sodium selenite 10 ng/mL, ovalbumin 100 μg/mL, and fibronectin 5 μg/mL were cultured in F12/DMEM medium.

Cells were cultured in a 96-well plate at 0.5 x 10 ⁴ /well, and hFGF11b and hFGF11b-delC were cultured together with heparin (10 μg/mL) at a concentration of 3 to 3000 ng/mL for 42 hours. The increase in cell number was confirmed by a method using WST-8 and a live imaging analysis method.

First, using WST-8[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt], an electron mediator ( electron mediator) and intracellular dehydrogenases (dehydrogenases) was confirmed by measuring the production level of WST-8 formazan (formazan). The degree of WST-8 formazan production can be confirmed through absorbance (450 nm).

The result is illustrated in FIG. 4 . Referring to FIG. 4 , cell proliferation activity was confirmed in both hFGF11b and hFGF11b-delC. In particular, it was confirmed that hFGF11b-delC exhibited a higher cell proliferative activity than hFGF11b.

Subsequently, changes in cell proliferation rates due to hFGF11b and hFGF11b-delC were confirmed using in vivo imaging equipment (Lionheart FX, BioTeK, VT, USA). It was checked at 2 hour intervals for 42 hours.

The result is illustrated in FIG. 5 . Referring to FIG. 5 , cell proliferation activity was confirmed in both hFGF11b and hFGF11b-delC. In particular, it was confirmed that hFGF11b-delC exhibited a higher cell proliferative activity than hFGF11b.

Although various embodiments have been described above, the scope of the rights is not limited thereto. The implemented form can be implemented with various modifications within the scope of the detailed description and accompanying drawings of the invention, and it is natural that this also falls within the scope of rights.

It can be applied to cosmetics and pharmaceuticals that use cell growth factors as main ingredients.

Claims (2)

1. A polypeptide having at least 90% identity over the entire length of the sequence of SEQ ID NO:2.
2. The polypeptide of claim 1, wherein the polypeptide of SEQ ID NO:2.

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