KR20200132276A - Method for Separating Sericin Protein for Preventing or Treating Bone Diseases and Uses Thereof - Google Patents

Abstract

The present invention relates to a method for separating a sericin protein and uses of the sericin protein separated by the same. Through a combination treatment of extraction by ultrasonic treatment and separation method according to molecular weight under specific temperature conditions, which is the method of the present invention, the sericin protein having a molecular weight of 30 kDa or more, which is a sericin-1/-3 protein fraction separated from silk, is a protein derived from a natural substance, thereby being safe, having less denaturation, being biocompatible, and also having an excellent bone regeneration effect. Therefore, the sericin protein having the molecular weight of 30 kDa or more, which is the sericin-1/-3 protein fraction separated from silk according to the method of the present invention, can be an excellent alternative to existing bone disease/injury treatment-related agents, and thus can contribute to economic growth and social benefits by recycling sericin, which is discarded as a by-product of silk protein.

Description

Method for Separating Sericin Protein for Preventing or Treating Bone Diseases and Uses Thereof}

The present invention relates to a method for isolating sericin protein for preventing and treating bone diseases, and to a use thereof.

In addition to the mechanical functions that support the human body and perform movement, bones play a role in regulating the calcium ion concentration in the body, and play an important physiological function in the bone marrow to produce red blood cells and white blood cells necessary for the human body. Bone consists of cortical bone, a dense structure with high mechanical strength, and strut bone, a structure like an internal sponge. As age increases, bone density decreases, causing fractures easily even with small impacts, and is damaged by osteoporosis and arthritis, and bone regeneration becomes difficult when fractures occur due to such physiological and physical damage.

In addition, the treatment methods for bone regeneration include natural treatments, drugs or physical therapy, orthopedic surgery, and if recovery is difficult due to a large degree of damage to the bone, normal tissues are protected and Remove damaged tissue.

Recently, attempts have been made to replace or regenerate damaged bone tissue with normal bone tissue, and accordingly, interest in the development of bone tissue or a bone regeneration promoting agent thereof for intracorporeal transplantation is increasing.

Accordingly, research is underway to develop artificial bones of biological tissues to increase the effect of bone formation by using cells such as bone marrow-derived stem cells to compensate for damaged bone tissues, and methods of transplanting bones from other people or animals or by patients themselves. There is also a method of transplanting the tissue of the patient, but there is a problem such as insufficient materials that can be used in the patient's body because an immunological rejection reaction occurs or the damaged area is large by transplanting another tissue.

Meanwhile, silk usually refers to a fibrous protein material obtained from silkworms, and has been widely used as a material for clothing, and research to use it as a new natural polymer resource is in progress. Since the composition of silk protein is very similar to the amino acid composition of collagen protein that makes up the human skin, it is used in food and cosmetics materials based on skin affinity. Bioengineering materials such as enzyme-immobilized carriers, cell culture supports, wound covering materials, artificial blood vessels, etc. It is used as a medical material.

These silks can be classified into two types of proteins, differing in the degree of orientation of the inner and outer layers of the fiber, the content of amino acids, molecular weight, and crystallinity. The ratio is approximately 25:75, which is defined as sericin and fibroin, and sericin has high solubility and is generally removed in the refining process, leaving only fibroin. This is generally called silk and is mainly used for clothing. In the past, when silk was used only for clothing, sericin was discarded as wastewater during the refining process, but recently, sericin has excellent biocompatibility and can be used as a biomaterial, and many studies have been conducted. As a method of separating sericin from silk, high-temperature treatment is mainly used in an alkaline solution, or water is added and treated at high temperature and high pressure for a long time.

Sericin varies in amino acid content and solubility from the outer layer to the inner layer in a living or dead state. Due to these characteristics, several scholars have distinguished types of sericin.

Watanabe (1959) divides sericin into A and B according to the solubility of sericin, and Komatsu (1966) divides sericin into Ⅰ, Ⅱ, and Ⅲ, and the weight ratio is 4:4:2. From this, sericin Ⅰ, Ⅱ, Ⅲ, Ⅳ is called sericin Ⅰ, Ⅱ, Ⅲ, Ⅳ, and sericin Ⅰ, Ⅱ is called soluble sericin, and some of sericin Ⅱ and Ⅲ, Ⅳ are classified as poorly soluble. Is that it is low.

Sericin Ⅰ, Ⅱ has many hydrophilic amino acid groups, and it is located in the outermost layer when the silkworm sediments, so it is in the position where moisture evaporation is the fastest, so it is easy to achieve amorphousness due to poor bonding strength such as hydrogen bonds between molecules due to the large number of pores. As it goes inside, the content of hydrophilic amino acids is lower than that of the outside, and the properties of fibroin become closer. There is a little wax layer between the sericin IV part and fibroin to protect fibroin, and sericin and fibroin can be distinguished by this boundary.

As a component of silk, sericin, which is generated as a by-product during refining, is a natural ingredient, but unlike fibroin, which has been recognized for its value as a fiber, various functional foods, and pharmaceutical uses, it cannot be used for various purposes and is disposed of as a waste through a separate process. It is a situation.

Therefore, in order to compensate for these problems and restore bone damage, replacement therapy using active ingredients of natural products that are easy to supply and are biocompatible and safe is required in reality, and a sericin protein of an appropriate size can help bone regeneration. In addition to being an alternative as a natural product, sericin, which is discarded as a by-product of silk protein, could be recycled, contributing to significant economic growth and social benefits.

The present inventors have made intensive research efforts to develop a material that can promote bone regeneration and can be safely applied to the human body without side effects. As a result, a sericin protein having a molecular weight of 30 kDa or more, which is a sericin-1/-3 protein fraction separated from silk, was obtained through extraction by ultrasonic treatment and a combination treatment according to molecular weight under specific temperature conditions, and the 30kDa sericin The protein promotes bone regeneration by up-regulating the expression of BMP (bone morphogenic protein), which is known to increase bone regeneration in macrophage cell lines. In addition, when the protein is filled in the bone defect site in the bone damage model, the bone damage and defect site are quickly recovered By confirming the reproduction, the present invention was completed.

Accordingly, an object of the present invention is to provide a method for producing a sericin protein derived from silk.

In addition, another object of the present invention is to provide a method for producing a therapeutic agent for bone regeneration comprising a sericin protein derived from silk.

In addition, another object of the present invention is to provide a pharmaceutical composition for preventing and treating bone diseases comprising a sericin protein derived from silk as an active ingredient.

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

Hereinafter, the present invention will be described in detail.

According to an aspect of the present invention, the present invention comprises the steps of: (a) subjecting a cocoon or a flat dog to ultrasonic waves to obtain a water-soluble silk protein fraction; And (b) separating and obtaining a sericin protein having an average molecular weight of 30 kDa or more from the fractionated protein fraction of step (a). It provides a method for producing a sericin protein derived from silk.

The sericin protein of the present invention is derived from silk.

In the present specification, the term "silk" refers to a fiber made by sediment of silkworm insects, and preferably means a gajamsa which is sedimented by silkworm ( Bombyx mori ). Silk protein consists of fibroin and sericin, and fibroin and sericin are present in an average of 75% and 25%, respectively.

As used herein, the term "sericin" refers to a high molecular weight protein composed of 18 amino acids and a broad molecular weight range from 10 kDa to 300 kDa. Sericin changes in amino acid content and solubility from the outer layer to the inner layer in a live or dead state. According to this solubility, sericin is divided into 4 stages and divided into sericin Ⅰ, Ⅱ, Ⅲ, Ⅳ from the outer layer. However, its fundamental characteristic is that the solubility is lower as it goes to the inner layer. Sericin Ⅰ, Ⅱ has many hydrophilic amino acid groups, and it is located in the outermost layer when the silkworm sediments, so it is in the position where moisture evaporation is the fastest, so it is easy to achieve amorphousness due to poor bonding strength such as hydrogen bonds between molecules due to the large number of pores. As it goes inside, the content of hydrophilic amino acids is lower than that of the outside, and the properties of fibroin become closer. There is a little wax layer between the sericin IV part and fibroin to protect fibroin, and sericin and fibroin can be distinguished by this boundary.

According to a preferred embodiment of the present invention, the sericin protein fraction with an average molecular weight of 30 kDa or more increases the expression of BMP (Bone morphogenic protein)-2 and BMP (Bone morphogenic protein)-4. Particularly, the sericin protein fraction having an average molecular weight of 30 kDa or more of the present invention exhibits a remarkably excellent effect of enhancing the expression of BMP-2 and BMP-4 compared to the sericin protein fraction having an average molecular weight of less than 30 kDa.

Further, according to a preferred embodiment of the present invention, the sericin protein is a sericin-1 fragment and a sericin-3 protein fragment.

In addition, according to a preferred embodiment of the present invention, the ultrasonic treatment in step (a) is performed at a temperature of 30°C to 70°C, preferably 65°C, but is not limited thereto as long as the target protein can be separated. .

In addition, according to a preferred embodiment of the present invention, the ultrasonic treatment in step (b) is performed with an ultrasonic wave of 0.1 to 1000 KHz, preferably of 10 to 50 KHz, and more preferably of 40 KHz. As long as a fraction of the protein can be extracted, it is not limited thereto.

According to a preferred embodiment of the present invention, the sericin protein of the present invention is prepared by extracting sericin from silk using ultrasonic waves and fractionating according to molecular weight.

In addition, the term "sericin protein" used herein while referring to a protein of interest refers to a sericin peptide fractionated according to molecular weight after sericin is extracted from silk using ultrasonic waves, and is used interchangeably with it. In addition, the sericin protein may include any natural or synthetic peptide as long as it has the physiological activity of sericin derived from silk.

Sericin-1 and sericin-3 protein fragments having an average molecular weight of 30 kDa or more, which are sericin proteins of the present invention prepared by the above method, are derived from natural proteins and are basically safe, and there is no high-temperature treatment step, so there is almost no denaturation of the protein, In addition to being biocompatible, it is selected through an efficacy test to enhance the expression level of BMP, which is known to increase bone regeneration and has excellent functionality.

According to another aspect of the present invention, the present invention comprises the steps of: (a) treating a cocoon or a plain spun silk mat with ultrasonic waves to obtain a water-soluble silk protein fraction; And (b) separating and obtaining a sericin protein having an average molecular weight of 30 kDa or more from the fractionated protein fraction of the (a) step, providing a method of producing a therapeutic agent for bone regeneration comprising a sericin protein.

The sericin protein included in the therapeutic agent for bone regeneration of the present invention is a protein produced by decomposing silk protein, a macromolecule, and has an effect of increasing the expression of BMP (Bone morphogenic protein)-2 and BMP (Bone morphogenic protein)-4. As long as it is exerted, it may be a sericin protein having an average molecular weight of 30 kDa or more, preferably a sericin protein having an average molecular weight of 30 kDa to 300 kDa, more preferably a sericin protein having an average molecular weight of 30 kDa to 50 kDa.

The therapeutic agent for bone regeneration of the present invention can promote bone remodeling and mineralization, and quickly regenerate bone damage and defects when filled in bone defects.

As used herein, the term "bone regeneration" or "bone regeneration" refers to a process in which bone is destroyed and absorbed by osteoclasts and then bone is re-formed by osteoblasts. The term "mineralization of bone" refers to a phenomenon in which minerals such as calcium are deposited on bone cells or extracellular matrix.

The therapeutic agent for bone regeneration of the present invention not only increases the expression of BMP (Bone morphogenic protein)-2 and BMP (Bone morphogenic protein)-4, but also affects bone cells by acting on the damaged bone region with a sericin protein having excellent biocompatibility. It can prevent the loss of bone cells. Therefore, it can be applied to manufacture bone cement with suitable physical properties depending on the purpose.

In addition, the therapeutic agent for bone regeneration of the present invention may additionally include at least one selected from the group consisting of biocompatible binders, antibiotics, vitamins, glucosamine, cytokines, and growth factors.

The biocompatible binder is from the group consisting of gelatin, hyaluronic acid, hydroxyapatite, collagen, fibrin, fibrinogen, thrombin, mussel adhesion protein, elastin, casein, albumin, keratin, chitin and chitosan. It may be one or more selected, but is not limited thereto.

In addition, the biocompatible binder is starch, polylactic acid, polyglycolic acid, polylactic-co-glycolic acid, polydioxanone, polycaprolactone, polycarbonate, polyoxoester, polyamino acid, polyanhydride, polyhydroxybutyl Rate, polyhydroxyvalyrate, poly(propylene glycol-co-fumaric acid), tyrosine-polycarbonate, polyvinylpyrrolidone, cellulose, ethyl cellulose, and carboxy methyl cellulose may be one or more selected from the group consisting of, It is not limited thereto.

In addition, the therapeutic agent for bone regeneration of the present invention can be injected directly into a bone defect site or filled with a bioinjectable graft material to achieve the desired effect.

In one embodiment of the present invention, it was observed that the level of the BMP protein was significantly higher when the sericin protein of the present invention was treated with a gelatin sponge to be transplanted into a bone injury model. Through this result, it can be seen that the sericin protein filled in the microporous structure of the gelatin sponge is dissolved by the biological fluid, and the bone regeneration efficacy is improved by maintaining a high BMP level by interacting with new biological bones generated from adjacent bone tissue.

In addition, the present invention can provide a bone graft comprising a sericin protein.

Sericin protein, an active ingredient of the present invention, may be included in a concentration of 0.01-1000 μg/ml with respect to the bone graft material, more preferably 0.1-600 μg/ml, and even more preferably 0.5-300 μg/ml , Most preferably 1-100 µg/ml.

Sericin protein, which is an active ingredient of the present invention, may be included in an amount of 0.01-40% by weight, more preferably 0.1-30% by weight, and most preferably 1-20% by weight based on the total weight of the bone graft material.

The term "bone graft material" as used herein refers to a graft material that is used to fill a space in bone tissue by replacing a part of the bone tissue due to various diseases or accidents, and to promote bone growth. It is defined as meaning.

The bone graft material is selected from the group consisting of growth factors promoting bone growth, fibrin, bone morphogenetic factors, bone growth agents, chemotherapeutic agents, antibiotics, analgesics, bisphosphonates, strontum salts, fluoride salts, magnesium salts, and sodium salts. It may additionally contain a biologically active substance.

The growth factors include platelet-derived growth factor (PDGF), transgenic growth factor (TGF-beta), insulin-like growth factor (IGF-I), IGF-II, fibroblast growth factor (FGF), and beta-II (beta). -2-microglobulin), etc. can be used. As the bone tissue formation enhancing peptide and protein, various peptides including an RGD sequence and various proteins such as collagen and fibronogen may be used. As the bone morphogenetic factor, osteocalcin, bonesialo protein, or osteogenin may be used. The bone growth agent may be used without limitation as long as it is harmless to the human body and a substance that promotes bone growth, and a nucleic acid that promotes bone formation, an antagonist of a substance that inhibits bone formation, and the like may be used.

According to another preferred embodiment of the present invention, as a compound for formulating the bone graft material of the present invention, hyaluronic acid, hydroxyapatite, collagen, calcium carbonate, calcium phosphate, calcium sulfate, monocalcium phosphate , A compound selected from the group consisting of tricalcium phosphate and silica, and most preferably hydroxyapatite.

In particular, hydroxyapatite is the same ingredient as the enamel of human bones and teeth, and has a great effect on restoring defects in the enamel quality of teeth. Because of this restoration effect, it restores the original color of the teeth. It is also in the limelight.

The bone graft material of the present invention can be molded and used in the form of putty, paste, moldable strips, blocks, chips, etc. using methods such as compression, compression, pressure contact, packing, compression, and hardening, and the compound is used. Therefore, it can be formulated and used in the form of gel, powder, paste, tablet, pellet, etc., and it is also possible to use as it is in powder form.

The bone region to which the bone graft material of the present invention can be applied is not particularly limited.

Accordingly, the sericin protein of the present invention alone or a therapeutic agent for bone regeneration containing the sericin protein of the present invention and an appropriate biocompatible binding agent, antibiotic, vitamin, glucosamine, cytokine and/or growth factor, is directly injected into the bone defect site, or In the case of filling and injection into a bioinjectable type of graft material, the effect of bone regeneration at the site of damage or defect of the bone is excellent.

Based on the above, the therapeutic agent for bone regeneration of the present invention can be used for various fractures, osteonecrosis diseases, or bone regeneration.

In addition, according to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing and treating bone diseases comprising the sericin protein as an active ingredient.

The bone disease may be one selected from the group consisting of osteoporosis, osteomalacia, rickets, fibrotic osteopathy, amorphous bone disease, metabolic bone disease, and bone damage, and as long as the bone regeneration effect is exhibited, it is not limited thereto.

The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The term "pharmaceutically acceptable carrier" as used herein is commonly used in formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It does not become.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity of the patient, Usually, the skilled practitioner can readily determine and prescribe the dosage effective for the desired treatment or prophylaxis.

According to one embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.0001-1000 mg/kg.

The pharmaceutical composition of the present invention is prepared by formulating a pharmaceutically effective amount using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art. It may be prepared in a dosage form or may be prepared by placing it in a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

As used herein, the term "therapeutically effective amount" means that the amount of an inhibitor to be administered alleviates one or more symptoms of bone disease, which is a disease to be treated, to some extent. Thus, a pharmaceutically effective amount means (1) reversing the rate of progression of bone disease or (2) stopping further progression of bone disease to some extent, and (3) one or more symptoms associated with bone disease It means an amount that has the effect of reducing (preferably, removing) to some extent.

Since the pharmaceutical composition of the present invention contains the above-described sericin protein as an active ingredient, descriptions of the contents in common between the two are omitted in order to avoid undue complexity in the present specification.

Sericin protein having a molecular weight of 30 kDa or more, which is a sericin-1/-3 protein fraction separated from silk, is a natural substance through a combination treatment of extraction by ultrasonic treatment under a specific temperature condition, which is the method of the present invention, and separation method according to molecular weight. As it is a protein derived from, it is safe, less denatured, biocompatible, and has excellent bone regeneration effect. Therefore, sericin protein having a molecular weight of 30 kDa or more, which is a sericin-1/-3 protein fraction isolated from silk according to the method of the present invention, can be an excellent alternative to the conventional bone disease/injury treatment-related preparations. In addition, sericin, which is discarded as a by-product of silk protein, can be recycled to contribute to economic growth and social benefits.

1 shows a schematic protein extraction method. 1A is a method for extracting sericin protein by a conventional hot water extraction method; Figure 1b is a method for extracting sericin protein from a cocoon or flat dog by ultrasonic treatment and molecular weight separation at 37 degrees Celsius; And FIG. 1C schematically shows a method for extracting sericin protein from a cocoon or flat dog by ultrasonic treatment and molecular weight separation at 65°C. Compared to the cocoon, flat dogs have the advantage of increasing the yield when sericin is extracted under the same conditions because the sericin content is 2-3 times higher. Not only disappears, but also because the protein is denatured, the ability of the isolated sericin protein to induce bone formation promoting proteins such as BMP is significantly lowered. Therefore, it can be seen that the method as shown in FIG. 1C is the most efficient under conditions of maximizing the yield of sericin and denaturing and removing proteins that are not conducive to bone regeneration.
Figure 2 shows two-dimensional (2D) electrophoresis (Figures 2A-2C) and Western blot results for BMP-2/-4 (Figures 2D-2F):
(A) 2D electrophoresis results and (D) Western blot results of products by conventional refining methods; (B) 2D electrophoresis results and (E) Western blot results of products from cocoons; (C) 2D electrophoresis results and (F) Western blot results of products from flat dogs.
At this time, the marked spots performed MALDI-TOF for identification, and in the case of (B), 10 spots were obtained, and the results are shown in Table 1; In the case of (C), three spots were obtained, and the results are shown in Table 2.
(D) The protein of the refining product showed a very weak increase in BMP-2/-4.
(E) The two fractions of (B) were divided by molecular weight (30 kDa). The high molecular weight fraction (average molecular weight of 30 kDa or more) showed a higher level of BMP-2/-4 increase than the low molecular weight fraction (average molecular weight of less than 30 kDa).
(F) Higher molecular weight fractions (average molecular weight less than 30 kDa) in flat dogs (average molecular weight of 30 kDa or more) showed increased levels of BMP-2/-4 expression.
3 shows the results of micro CT. The gelatin sponge group containing the sericin fraction (Gelatin with sericin) showed a higher bone regeneration rate compared to the unfilled control or the gelatin-only sponge group (Gelatin only). When comparing the amount of bone (BV; bone volume), the difference between groups showed a statistically significant difference (P <0.001). In the post hoc test, the difference between the gelatin sponge group containing the sericin fraction and the other groups was statistically significant (* In the case of the control group without gelatin sponge and the gelatin-only sponge group *P <0.001 and = 0.015 ).
4 shows the histological results. New bone formation was prominent in the group of gelatin sponges containing the sericin fraction. The immunostaining results of BMP-2/-4 showed higher expression in the gelatin sponge group containing the sericin fraction of the present invention compared to other groups.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and that the scope of the present invention is not limited by these examples according to the gist of the present invention, for those of ordinary skill in the art to which the present invention belongs. It will be self-evident.

Example

Experimental materials and methods

Cell culture and silk protein processing

RAW264.7 mouse macrophages (Korea cell line bank number 40071) were cultured in a culture medium.

As a comparative and control experimental method, sericin 1 extracted by heating a cocoon in distilled water as a product by the conventional hot water extraction method was used.

As another comparative and control experimental method, a soluble silk protein fraction was extracted from the inner layer by applying ultrasonic waves (40 KHz) to a cocoon in distilled water at 37°C.

In addition, as a method of the present invention, a soluble protein silk fraction was extracted by treating a plain spun silk with ultrasound (40 KHz) in distilled water at 65°C. The extracted silk protein was separated by a filter (Microcon®-30, Merk Millipore Ltd., Tullagreen, Ireland). Filtering was performed according to the manufacturer's protocol.

In flat dogs, 1/3 of sericin is present in a higher proportion than that of conventional cocoons. This was separated according to the molecular weight, and a fraction having a molecular weight of 30 kDa or more was used for later experiments.

RAW264.7 cells were dispensed into a 6-well culture plate, and treated with 1, 5 and 10 μg/mL of the scouring product or the soluble silk protein fraction isolated by the above method. After 2, 8 or 24 hours of incubation, cells were obtained. Cells cultured as a control were treated with the same amount of solvent as required for sericin.

Sample preparation and 2-DE

Samples were suspended in 7M urea, 2M thiourea, 4% (w/v) CHAPS, 2.5% (w/v) DTT and a protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN, USA). The lysate was homogenized and centrifuged at 15,000 x g for 20 minutes and then stored at -80°C. Protein concentration was determined by the Bradford method (Bio-Rad, Hercules, CA, USA). For 2-DE analysis, pH 4-7 IPG strips in expansion buffer containing 7M urea, 2M thiourea, 2.5% (w/v) DTT and 4% CHAPS (GE Healthcare Life Sciences, Pittsburgh, PA, USA) Was rehydrated. The protein lysate (600 μg) was loaded onto the rehydrated IPG strip using IPGphor III (GE Healthcare Life Sciences, Pittsburgh, PA, USA), and 2 on 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel). D separation was carried out. After fixing the gel in a 40% (v/v) methanol solution containing 5% (v/v) phosphoric acid for 1 hour, the gel was used as a colloidal Coomassie blue G-250 solution (ProteomeTech, Seoul, Korea). Dyed. The gel was destained using deionized water and images were acquired with an image scanner (Bio-Rad, Hercules, CA, USA). Image analysis was performed using ImageMasterTM 2D Platinum software (Amersham Biosciences, Hercules, CA, USA). For comparison of protein spots, more than 25 spots were standardized by landmarking on all gels.

Digestion and protein extraction in gels using trypsin

The protein band was cut out from the SDS-PAGE gel and the gel was digested with trypsin according to the existing procedure. In summary, protein bands were cut from stained gels and cut into pieces. The gel pieces were washed with 25 mM ammonium bicarbonate buffer (pH 7.8) containing 50% (v/v) acetonitrile (ACN) for 1 hour at room temperature. After dehydrating the gel pieces in a centrifugal concentrator (Biotron, Inc., Incheon, Korea) for 10 minutes, the gel pieces were rehydrated in 50 ng of sequencing grade trypsin solution (Promega, Madison, WI, USA). After incubation with 25 mM ammonium bicarbonate buffer (pH 7.8) overnight at 37°C, sonication with 5 µl of 0.5% formic acid (FA) containing 50% (v/v) ACN for 40 minutes to digest by trypsin The peptide was extracted. The extracted solution was concentrated using a centrifugal concentrator. Prior to mass spectrometry analysis, the peptide solution was desalted using a reverse phase column. Briefly, after the equilibration step with 10 μl of 5% (v/v) formic acid, the peptide solution was loaded onto the column and washed with 10 μl of 5% (v/v) formic acid. The bound peptide was eluted with 5 µl of 70% ACN with 5% (v/v) formic acid.

Identification of proteins by LC-MS/MS

LC-MS/MS analysis was performed by ACQUITY UPLC and LTQ-orbitrap-mass spectrometer (Thermo Electron, San Jose, CA). The column was a BEH C18 1.7 μm, 100 μm x 100 mm column (Waters, Milford, MA, USA). Mobile phase A for LC separation was 0.1% formic acid in deionized water, and mobile phase B was 0.1% formic acid in acetonitrile. The chromatographic gradient was set to increase linearly from 10% B to 40% B for 21 minutes, 40% B to 95% B for 7 minutes, and 90% B to 10% B for 10 minutes. The flow rate was 0.5 μL/min. For tandem mass spectrometry, mass spectra were acquired using data-dependent acquisition with MS/MS scan after full mass scan (300-2000 m/z). Each MS/MS scan acquired was an average of 1 microscan in LTQ. The temperature of the ion transfer tube was adjusted to 275°C and the spray was 1.5-2.0 kV. The normalized collision energy was set to 35% for MS/MS. Individual spectra of MS/MS were processed using SEQUEST software (Thermo Quest, San Jose, CA, USA), and the resulting peak list was queried in a house database using the MASCOT program (Matrix Science Ltd., London, UK). Used to. We set methionine, modification of cysteine, methylation of arginine, phosphorylation of serine, threonine and tyrosine for MS analysis, and the tolerance of peptide mass was 10 ppm. The MS/MS ion mass tolerance was 0.8 Da and the cut-off tolerance was 1, and the state of charge (+2, +3) was considered for data analysis. We only took significant hits defined by MASCOT probability analysis.

Western Blotting

In order to analyze the expression of BMP-2/-4 induced by the soluble silk protein fraction isolated by the method of the present invention, the cells were treated with 1, 5, and 10 μg/mL of the silk protein fraction. Protein was obtained and mixed with SDS buffer. After thermal denaturation, electrophoresis was performed on a 10% polyacrylamide gel. The gel was transferred to a polyvinylidene difluoride membrane. After blocking, the membrane was probed with a primary antibody (BMP-2/-4, Santa Cruz Biotech) (dilution ratio=1:500). Blots were imaged and quantified using the ChemiDoc XRS system (Bio-Rad Laboratories).

Animal experiment design

In this example, 8-week-old Crl:CD (Sprague-Dawley) SPF (specific pathogen-free)/VAF rats (Orientbio Inc., Sungnam, Korea) were used. All procedures were performed in accordance with the laboratory animal care guidelines and approved by Gangneung-Wonju University Animal Testing Laboratory (GWNU-2018-14).

Before the experiment, 22 rats (2-3 rats per cage) were reared under a 12-hour light-dark cycle for a week in an environment controlled at 20-22° C. and 40% humidity. Rats were free to consume food and water and were fed a controlled semi-synthetic diet (74% carbohydrates in soybean vegetable oil, 14% protein in casein, supplemented with a standard vitamin and mineral mixture) according to conventional recommendations.

To construct a cranial tube defect model, 0.4 mL ketamine (100 mg/mL; Ketara; Yuhan, Seoul, Korea) and 0.3 mL xylazine (10 mg/kg body weight; Rompun; Bayer Korea, Seoul, Republic of Korea) was administered intramuscularly to rats to induce general anesthesia. The rat's skull was depilated and sterilized with povidine-iodine. The skull was incised longitudinally from the nasal bone to the occipital protuberance, and a midline incision was made in the periosteum. A sharp subperiosteal incision was made to reveal the skull membrane to expose the parietal bone. While flowing a large amount of saline, a dental-trephine bur (diameter: 8.0 mm) was used to induce full-thickness of the skull in both areas. Two 9 mm thick severe damages were created, one on each side of the midline.

Rats were divided into three groups. In consideration of the high mortality rate of cranial tube defect surgery, additional rats were assigned to each group. The graft material consisted of a gelatin sponge (Cutanplast Dental®, Uniplex, Sheffield, UK) or a gelatin sponge (Cutanplast Dental®) containing sericin-1/3 isolated according to the method of the present invention. The amount of sericin-1/-3 in each graft was about 50 μg. Group 1 (control) consisted of 6 animals that received anesthesia and incision, but no grafts. Group 2 was treated with a gelatin sponge, and Group 3 was treated with a gelatin sponge containing sericin. Group 2 had 6 animals and group 3 had 10 animals. After the graft was transplanted, the skin and periosteum were simultaneously sutured with 3-0 black silk. After surgery, antibiotics and analgesics were administered. At the first 24 o'clock, 1 in group 2 and 2 in group 3 died. Therefore, 6 animals in group 1, 5 animals in group 2, and 8 animals in group 3 were followed up to 8 weeks after surgery. After 8 weeks, all mice were sacrificed and micro-CT and histological analysis were performed.

Micro-computed tomography

Cranial tube samples (10 x 10 x 0.5 mm) were sent to the Korean Institute of Basic Science (Ochang, Korea) for micro-CT analysis. The subsequent procedure was carried out according to the description of the previous publication. Briefly, an animal PET/CT/SPECT system (Inveon Siemens, Malvern, PA, USA) was used for analysis. Scanned images were reconstructed using the supplied software (Inveon Siemens). The region of interest (ROI) was determined based on the size of the initial surgical defect. The amount of bone (BV) in the ROI was calculated by setting a threshold level of 25% bone reference.

Immunohistochemical confirmation and Western blot analysis in tissue samples

In order to evaluate the expression of BMP-2/-4, immunohistochemical staining was performed using an anti-BMP-2/-4 antibody (CAT #: sc-137087, Santa Cruz Biotech). Procedures for immunohistochemical analysis are disclosed in previous publications. Briefly, sections were prepared and enzymatically digested using proteolytic enzyme (1 mg porcine trypsin, Sigma-Aldrich). Then, the sections were treated with hydrogen peroxide. After washing and blocking steps, the sections were treated with the primary antibody (BMP-2/-4, 1:100). After conjugation with a universal secondary antibody (Dako REAL™ EnVision™/HRP, rabbit/mouse, Dako North America Inc.), the slides are diaminobenzidine chromogen and hydrogen peroxidase (Dako REAL™ DAB+ Chromogen and Dako REAL™. Substrate buffer; Dako North America Inc.).

Example 1. BMP upregulation of silk-derived sericin protein

Cocoon or silk is largely composed of two substances: fibroin and sericin. Among them, the process of removing sericin surrounding fibroin is called refining. Conventional refining methods include a method of boiling Marseille soap and sodium carbonate in an alkaline aqueous solution, such as a refining method using a proteolytic enzyme such as asparse, or a high-temperature high-pressure method using a high-temperature high-pressure cooker. In the refining method, fibroin from which sericin has been removed is used as fiber, and the remaining sericin-containing liquid is discarded as wastewater.

In addition, in order to extract sericin from wastewater, considerable technology and effort are required to separate sericin and other substances, and the efficiency is low economically. Therefore, the general alkaline refining method of silk is used to recover pure sericin. It is not easy, and it is difficult to control molecular weight and dissolve poorly soluble sericin, so that the water-soluble sericin collection rate is low, and treatment reagents have a problem that it affects sericin, a protein, as well.

In addition, in the case of the conventional hot water extraction method, there is a disadvantage in that the protein is denatured by high temperature.

Accordingly, the inventors of the present invention solved the above-described problems, and obtained a sericin protein fraction through the combination treatment of the present invention, and among them, the ability to induce BMP-2/4 is excellent in high molecular weight (average molecular weight of 30 kDa or more). As a novel sericin protein extraction method capable of obtaining only the minimally denatured sericin protein, a method in which an extraction method by ultrasonic waves and a separation method according to molecular weight are combined while limiting the extraction temperature was constructed.

Accordingly, a silk-derived sericin protein in the form of a protein that was hardly denatured by heat was obtained by the method described in the'cell culture and silk protein treatment' method, and was confirmed through 2D electrophoresis results.

That is, 2D electrophoresis and Western blotting for BMP-2/4 were performed to analyze the results obtained through the above method.

As a result, as shown in Figs.2B and 2C, compared to the conventional method, the product refined only by the hot water extraction method without ultrasonic treatment is subjected to 2D electrophoresis (Fig.2A), the original extracted by ultrasonic treatment having a limited extraction temperature It can be seen that the soluble fraction of the invention shows much more protein spots. In addition, the protein fraction in Figure 2A was confirmed to be sericin 1.

In FIG. 2B, 10 spots were selected for the sericin protein obtained from the cocoon by sonication and separation according to molecular weight, and as a result of MALDI-TOF analysis for them, as shown in Table 1 below, most of them were protease inhibitors. Confirmed. In addition, the bands present at the positions of sericin 1 and sericin 3 were identified, but in addition to these, a number of other proteins were also present, so it was not possible to clearly confirm which bone formation-inducing protein was.

On the other hand, the number of spots in FIG. 2C for sericin protein obtained from plain spun silk by sonication and separation according to molecular weight was reduced to three spots, and the MALDI-TOF analysis results for them are shown in Table 2 below. Shown in.

That is, spot 1 of FIG. 2C was identified as a mixture of sericin 1 and sericin 3. Spot 3 in FIG. 2C was identified as sericin 3. It can be seen that spots 1 and 3 are fragments of sericin 1 and 3 proteins when considering the original molecular weight. Spot 2 was found to be keratin and is believed to be due to contamination.

Based on these results, it was confirmed that sericin proteins derived from silk proteins isolated according to the method of the present invention were sericin-1 and sericin-3.

In addition, the present inventors examined the ability to increase BMP-2/-4 expression by applying the extracted protein fraction to RAW264.7 cells as described above.

As a result, as shown in Figs. 2D to 2F, the protein of the refining product showed a very weak increase in BMP-2/-4 (Fig. 2D), whereas when the sonication product was separated according to the molecular weight, the fraction having a large molecular weight ( The average molecular weight of 30 kDa or more) showed a higher level of BMP-2/-4 increase than the low molecular weight fraction (average molecular weight of less than 30 kDa) (FIG. 2E). In addition, the protein fraction (average molecular weight of 30 kDa or more) of ordinary spun silk also increased the expression level of BMP-2/-4 (FIG. 2F).

That is, the high molecular weight sericin-1/-3 protein derived from silk isolated according to the method of the present invention was able to remarkably increase the expression of BMP-2/-4 when compared to the general refining product treatment. It is believed to promote bone formation.

number NCBI Blast Protein name Score Bell One NP_001139719.1 serine protease inhibitor 28 77 Bombyx mori 2 XP_004930020.1 serine/threonine-protein kinase 17 Bombyx mori 3 NP_000217.2 keratin 457 Homo sapiens 4 NP_000217.2 keratin 73 Homo sapiens 5 P00761.1 trypsin 55 Homo sapiens 6 XP_004930446.1 peptidyl-prolyl cis-trans isomerase H 22 Bombyx mori 7 XP_004930446.1 peptidyl-prolyl cis-trans isomerase H 16 Bombyx mori 8 NP_006112.3 keratin 180 Homo sapiens 9 NP_000412.3 keratin 281 Homo sapiens 10 NP_000412.3 keratin 147 Homo sapiens

number NCBI Blast Protein name Score Bell One NP_001108116.1 Sericin 3 161 Bombyx mori XP_012547623.1 Sericin 1 148 Bombyx mori 2 NP_000412.3 Keratin 85 Homo sapiens 3 NP_001108116.1 Sericin 3 151 Bombyx mori

Example 2. Bone regeneration effect by high molecular weight (average molecular weight of 30 kDa or more) sericin-1/-3 protein isolated according to the method of the present invention in a bone injury model

In order to demonstrate the bone regeneration effect of the sericin-1/-3 protein having a high molecular weight (average molecular weight of 30 kDa or more) identified in Example 1, the present inventors produced and processed a bone injury model.

As a result, as shown in FIG. 3, the gelatin sponge group containing the sericin fraction (Gelatin with sericin) had a higher bone regeneration rate compared to the unfilled control or gelatin-only sponge group (Gelatin only). Showed. Bone volume (BV; bone volume) was 0.51 ± 0.40 in the control group without gelatin sponge, respectively; Gelatin-3.13 ± 2.27 for the group of sole sponges; And in the case of the gelatin sponge group containing the sericin fraction was 8.48 ± 3.97 mm ³ . When comparing the amount of bone (BV), there was a statistically significant difference between groups (P <0.001). In addition, in the post-hoc test, the difference between the gelatin sponge group containing the sericin fraction and the other groups was statistically significant (P<0.001 and 0.015 in the control group without gelatin sponge and the gelatin-only sponge group). .

In addition, as shown in Figure 4, new bone formation was noticeable in the gelatin sponge group containing the sericin fraction, and the results of immunostaining of BMP-2/-4 were compared to the other groups by the sericin fraction isolated by the method of the present invention. It showed higher expression in the gelatin sponge group containing this.

In other words, when the high molecular weight sericin-1/-3 protein derived from silk isolated according to the method of the present invention is transplanted to a bone defect site of a bone damage model, it exhibits excellent bone regeneration effect and recovers bone damage and defects quickly. Made it.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments and are not intended to limit the scope of the present invention to those of ordinary skill in the art. Therefore, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (10)

(a) treating a cocoon or a plain spun silk mat with ultrasonic waves to obtain a water-soluble silk protein fraction; And
(b) separating and obtaining a sericin protein having an average molecular weight of 30 kDa or more from the fractionated protein fraction of step (a); comprising, a method for producing a sericin protein derived from silk.
The method of claim 1,
The sericin protein fraction having an average molecular weight of 30 kDa or more increases the expression of bone morphogenic protein (BMP)-2 and bone morphogenic protein (BMP)-4.
The method of claim 1,
The sericin protein is a sericin-1 protein fragment, a sericin-3 protein fragment, or a mixture thereof, characterized in that the sericin protein production method.
The method of claim 1,
The method for producing a sericin protein, characterized in that the ultrasonic treatment in step (a) is carried out at a temperature of 30°C to 70°C.
The method of claim 1,
The ultrasonic treatment in step (a) is characterized in that the ultrasonic treatment of 0.1 to 1000 KHz is carried out, sericin protein production method.
The method of claim 1,
The sericin protein having an average molecular weight of 30 kDa or more obtained by separation in step (b) is used for bone re-use.
(a) treating a cocoon or a plain spun silk mat with ultrasonic waves to obtain a water-soluble silk protein fraction; And
(b) a method for producing a therapeutic agent for bone regeneration comprising a sericin protein comprising the step of separating and obtaining a sericin protein having an average molecular weight of 30 kDa or more from the fractionated protein fraction of step (a).
The method of claim 7,
The bone regeneration therapeutic agent additionally comprises at least one selected from the group consisting of biocompatible binders, antibiotics, vitamins, glucosamine, cytokines, and growth factors, characterized in that the method for producing a therapeutic agent for bone regeneration comprising a sericin protein .
The method of claim 7,
The method for producing a therapeutic agent for bone regeneration comprising a sericin protein, characterized in that the therapeutic agent for bone regeneration can be injected directly into a bone defect site or filled in a bioinjectable type of graft material.
A pharmaceutical composition for preventing and treating bone diseases, comprising as an active ingredient a sericin protein derived from silk, prepared according to the manufacturing method of any one of claims 1 to 6.

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