KR101492169B1 - Electrospining nanofibers reinforced by mussel coating protein and their application - Google Patents

Abstract

The present invention relates to a scaffold for nanofiber tissue engineering comprising a mussel adhesive protein and an Fe ^& lt ^{; 3 + &} gt ^; ion and a process for their preparation.

Description

TECHNICAL FIELD The present invention relates to a nanofiber reinforced by mussel coating protein,

The present invention relates to a scaffold for nanofiber tissue engineering comprising a mussel adhesive protein and an Fe ^& lt ^{; 3 + &} gt ^; ion and a process for their preparation.

Tissue engineering technology refers to a technique of culturing cells on a support to prepare a cell-support complex and regenerating living tissues and organs using the same. Tissue engineering techniques are applied to the regeneration of almost all organs of human body such as artificial skin, artificial bone, artificial cartilage, artificial cornea, artificial blood vessel, artificial muscle. In order to optimize the regeneration of living tissues and organs in such tissue engineering techniques, it is important to prepare a tissue engineering support similar to a living tissue. The basic requirement of the tissue engineering support is that the tissue cells must adhere to the supporter and proliferate well, the function of the differentiated cells must be preserved, and even after transplantation into the body, they are compatible with surrounding tissues and become inflamed or clotted There must be non-toxic biocompatibility. In addition, when the transplanted cells function and function as new internal tissues, they should have biodegradability that can be completely decomposed and eliminated in a desired period of time.

Currently commonly used tissue engineering scaffolds are natural or synthetic polymer scaffolds which are natural and natural such as collagen, chitosan, gellatin, hyaluronic acid, alginic acid, Polymers, and synthetic polymers such as polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactic acid (PCL), and copolymer thereof. Such supports have been introduced as medical materials for supporters and cosmetics, wound dressings, and dental matrices for cell and tissue culture and implantation purposes. However, these materials have limited physical properties and thus have many limitations as materials for regeneration of human tissue organs that require various physical properties. In addition, it has been difficult from the past to sufficiently adhere, maintain and propagate physiologically active substances such as cells on the surface of a support. Therefore, it is necessary to develop a support for tissue engineering that can replace existing materials.

On the other hand, mussel, a marine creature, produces and secretes a byssus or byssal thread as a fiber material, so that the mussel itself can be firmly attached to a wet solid surface such as a rock in the sea, It is not affected by the effect. The mussel's footpath is coated with a very thin coating material, and the adhesive is released at the tip of the buttocks, helping to immobilize the mussel in the water. Especially, Mytilus specis mussel 's coating is similar to hard epoxy resin in mechanical properties (strength and hardness), but it can be used as a coating material to enhance the physical properties of various materials because the elongation is ~ 70% or more. Do.

In particular, in the case of the Meutilus edulis foot protein 1 (Mefp-1) protein, which constitutes the family of the mussel, dihydroxy-phenylalanine (DOPA) residues present in the protein bind to Fe ^{3 +} It is known that the hard coating layer formed by such bonding plays an important role in allowing the footbath to withstand external impacts. It is also known that DOPA residues are oxidized to DOPA-quinone, which binds to the surrounding DOPA residues and amino acids to cause crosslinking, which helps maintain strong binding even in water. In recent years, attempts have been made to apply gels or bioadhesives using DOPA-metal bonding and DOPA oxidation reaction. However, most of them are based on synthetic polymers, and there is no attempt to use gel or nanofibers using mussel adhesive protein and Fe ^{3 +} ions.

In a previous study, we developed a new type of mussel adhesive protein, fp-151, which was fused to both ends of Mgfp-5 by repeating the decapeptide repeated 80 times in Mefp-1 six times. The recombinant mussel It has been confirmed that the adhesive protein can be mass-produced in E. coli, and the purification process is also very simple, which is highly industrially applicable (WO2006 / 107183 or WO2005 / 092920).

Based on such mass production technology, the present inventors have succeeded in converting a tyrosine residue of a mussel adhesive protein mass-produced in an Escherichia coli system into DOPA with high efficiency and binding Fe ^{3 +} ions to a DOPA residue to produce nanofibers having enhanced mechanical properties . The nanofiber thus produced is very simple in its manufacturing process, and is highly industrially applicable and has excellent biocompatibility, which is suitable as a support for tissue engineering and a medical material.

One object of the present invention is to provide a nanofiber tissue engineering support comprising a mussel adhesive protein and Fe ^& lt ^{; 3 + &} gt ^; ions.

It is another object of the present invention to provide a method for producing the support for nanofiber tissue engineering.

In one aspect, the present invention relates to a nanofiber tissue engineering support comprising a mussel adhesive protein and an Fe ^& lt ^{; 3 + &} gt ^; ion.

In the present invention, "mussel adhesive protein" or "mussel coating protein" is an adhesive protein derived from mussel, preferably Mytilus edulis , Mytilus < RTI ID = 0.0 > galloprovincialis , or Mytilus coruscus , or a variant thereof.

For example, the mussel adhesive protein of the present invention is Mefp respectively derived from the species of mussel (Mytilus edulis foot protein) -1, Mgfp (Mytilus galloprovincialis foot protein) -1, Mcfp ( Mytilus coruscus foot protein) -1, 2-Mefp, Mefp-3, 3-Mgfp and Mgfp-5, or may comprise a variant thereof, preferably fp (foot protein) -1 (SEQ ID NO: 1), fp-2 (SEQ ID NO: 6), fp-3 (SEQ ID NO: 7), fp-4 (SEQ ID NO: 8), fp-5 (SEQ ID NO: 9), and fp- A fusion protein in which two or more kinds of proteins are linked, or a variant of the protein.

In addition, the mussel adhesive proteins of the present invention include all of the mussel adhesive proteins described in WO2006 / 107183 or WO2005 / 092920. Preferably, the mussel adhesive protein is selected from the group consisting of fp-151, fp-131, fp-353, fp-153, fp-351, 16). ≪ / RTI >

In addition, the mussel adhesive protein of the present invention may include a polypeptide in which the decapeptide (SEQ ID NO: 2) repeated about 80 times in fp-1 is continuously linked 1 to 12 times or more. Preferably, the decapeptide of SEQ ID NO: 2 is but is not limited to the fp-1 variant polypeptide (SEQ ID NO: 4) linked in 12 consecutive times.

In addition, in the present invention, the mussel adhesive protein may include an additional sequence at the carboxyl terminal or amino terminal of the mussel adhesive protein or some amino acid substituted with another amino acid under the premise of maintaining the adhesive property of the mussel adhesive protein. More preferably, a polypeptide consisting of 3 to 25 amino acids including RGD (Arg Gly Asp) is linked to the carboxyl terminal or amino terminal of the mussel adhesive protein, or a polypeptide consisting of 1 to 100 of the total number of tyrosine residues constituting the mussel adhesive protein %, Preferably 5 to 100% of which is substituted with 3,4-dihydroxyphenyl-L-alanine (DOPA).

RGD (Arg Gly Asp, SEQ ID NO: 17), RGDS (Arg Gly Asp Ser, SEQ ID NO: 18), RGDC (Arg Gly Asp Cys, SEQ ID NO: 19), RGDV (Arg Gly Asp Val, SEQ ID NO: 20), RGDSPASSKP (Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro, SEQ ID NO: 21), GRGDS (Gly Arg Gly Asp Ser, SEQ ID NO: SEQ ID NO: 23), GRGDSP (Gly Arg Gly Asp Ser Pro, SEQ ID NO: 24), GRGDSPC (Gly Arg Gly Asp Ser Pro Cys, SEQ ID NO: 25) and YRGDS (Tyr Arg Gly Asp Ser, No. 26) may be used.

Examples of mutants of the mussel adhesive protein to which the polypeptide consisting of 3 to 25 amino acids including RGD are linked at the carboxyl terminal or amino terminal of the mussel adhesive protein include, but are not limited to, the amino acid sequence of SEQ ID NO: 3 1 variant-RGD polypeptide consisting of the amino acid sequence of SEQ ID NO: 5 or the fp-151-RGD polypeptide consisting of the amino acid sequence of SEQ ID NO: 12, But is not limited thereto.

Preferably, the mussel adhesive protein of the present invention is modified such that at least 20% of all tyrosine residues are modified with DOPA. In addition, it is preferable to use a mussel adhesive protein which can be dissolved in an aqueous solution at an excellent solubility of 20 wt% or more. Tyrosine accounts for about 20-30% of the total amino acid sequence of most mussel adhesive proteins. Tyrosine in natural mussel adhesive proteins is converted to DOPA by the addition of OH groups through the hydration process. However, since the tyrosine residues of the mussel adhesive protein produced in Escherichia coli are not modified, it is desirable to carry out a modification reaction in which tyrosine is converted into DOPA by a separate enzyme and a chemical treatment method.

Methods for modifying the tyrosine residue contained in the mussel adhesive protein with DOPA can be used, and there is no particular limitation. As a preferred example, tyrosinase can be used to modify tyrosine residues to DOPA residues. In a specific example of the present invention, it has been shown that a mussel adhesive protein can be produced which satisfies the DOPA conversion and solubility conditions through a modification reaction using tyrosinase in vitro (FIG. 2).

The present invention provides a support for nanofiber tissue engineering based on mussel adhesive protein.

The natural extracellular matrix consists of a three-dimensional structure in which protein fibers with nanoscale dimensions are intertwined. Accordingly, the present invention uses nanofibers for preparing supports in order to realize a structure similar to natural extracellular matrix. Since the nanofibers have a large specific surface area and a large cell attachment area, when the cells are cultured on a support made of nanofibers, the cell adhesion is excellent.

In addition, the mussel adhesive protein can be mass-produced in E. coli, and the purification process is also very simple and highly industrially applicable. Further, conventionally, formaldehyde or glutaraldehyde was used as a crosslinking agent in order to enhance stability in water when producing nanofibers. Such crosslinking agents are unsuitable as supports for tissue engineering because they have toxicity to humans. In the present invention, DOPA existing in the mussel adhesive protein reacts with Fe ^{3 +} ions to form a bridge. DOPA and Fe ^{3 +} are advantageous in biocompatibility since they are substances already existing in the body.

In the present invention, various types of functional physiologically active substances can be easily coated on the surface of nanofibers prepared using mussel adhesive protein and Fe ^{3 +} , without physicochemical treatment, by using the adhesive force of mussel adhesive protein.

In the present invention, physiologically active substances that can be adhered to the nanofibers are collectively referred to as materials that promote the growth and differentiation of cells through interaction with cells or tissues of the human body, and also contribute to the regeneration and recovery of tissues. The physiologically active substance is collectively referred to as various biomolecules that can be included to realize an artificial extracellular matrix having a structure similar to a natural extracellular matrix.

Specifically, the physiologically active substance includes cells, proteins, nucleic acids, sugars, enzymes and the like, and examples thereof include cells, proteins, polypeptides, polysaccharides, monosaccharides, oligosaccharides, fatty acids and nucleic acids. Cells. The cells may be all cells including prokaryotes and eukaryotes. Examples of the cells include osteoblasts, fibroblasts, hepatocytes, neurons, cancer cells, B cells, An immune cell including a white blood cell, an embryo cell, and the like. In addition, the physiologically active substance includes, but is not limited to, a green fluorescent protein, a plasmid nucleic acid as a nucleic acid material, a hyaluronic acid, a heparin sulfate, a chondroitin sulfate, an alkaline salt as a sugar material, and an alkaline phosphatase as an enzymatic substance.

The nanofiber tissue engineering support of the present invention can provide a biomaterial-like support to optimize the regeneration of biological tissue and organs in tissue engineering techniques. In addition, the supporter of the present invention can be used to easily embody an extracellular matrix, and can also be used as a medical material for cosmetics, wound dressings, and dental matrix.

In addition, as one embodiment for providing the support for nanofiber tissue engineering,

(a) reacting mussel adhesive protein and Fe < ^{3 + &} gt ^; ions; And

(b) electrospun mixing the biodegradable polymer.

To a method for producing a nanofiber tissue engineering support.

The step (a) is a step of binding DOPA and Fe ^{3 +} ions contained in the mussel adhesive protein. DOPA is known to the mono-, bis-, tris- combination according to the pH and combined with a metal containing Fe ^{+ 3.} In addition, it is known that DOPA-metal bond has unique color depending on the bonding pattern. The color change due to the bonding number of DOPA-metal is well known by the existing studies.

In this method, Fe ^{3 +} can be used a reagent containing Fe ^{3 +} ions to ions and to provide, for example, but are not limited to, preferably one using the FeCl _3. More preferably, the mussel adhesive protein, DOPA in the solution is dissolved in 30 ~ 50wt%: the ratio of the Fe ^{3 +} molecule preferably 3: 1 or the addition of FeCl ₃ solution to reduce further the proportion of Fe ^{3 +} and , And the combination of DOPA and Fe ^{3 +} ions can be formed by raising the pH to 11 or more using NaOH.

The step (b) is a step of producing a three-dimensional nanofiber support, and it is preferable to use an electrospinning process.

The electrospinning process is a technique of forming fibers by utilizing the electrical attraction and the repulsive force generated when the polymer solution or molten polymer is charged to a predetermined voltage. The electrospinning process is capable of producing fibers with various diameters of several nanometers to several thousands of nanometers in size, has a simple equipment structure, can be applied to a wide range of materials, has a porosity higher than that of conventional fibers, Fiber fabrication is possible.

Specifically, in order to perform the electrospinning process, the mussel adhesive protein may be dissolved in an organic solvent alone or in a mixed solvent of an organic solvent and an acidic solvent. The organic solvent includes HFIP (hexafluoroisopropanol), HFP (hexafluoropropanol), TFA (trifluoroacetic acid) and the like, but is not limited thereto. In the present invention, it is preferable to mix HFIP at 100%. At this time, the concentration of the mussel adhesive protein dissolved in the solvent may be 10% (w / v) or more, preferably 30% (w / v). The spinning process can be carried out using the appropriate spinning device while applying the appropriate voltage. It is preferable to set the voltage applied during the spinning process within a range of 10 to 20 kV because a stable spinning process can be performed. When the voltage is increased within the voltage range of 10 to 20 kV, the spinning speed is increased.

Preferably, the mussel adhesive protein-Fe ^{3 +} is blended with the biodegradable polymer to prepare a synthetic polymer solution, and then nanofibers can be prepared through electrospinning.

The polymer generally includes most biodegradable polymers used as tissue engineering materials. For example, polycaprolactone (PCL), polydioxanone (PDO), poly (L-lactide) and PLLA (poly-DL-lactide-co-glycolide), which are known to dissolve in HFIP solvents, , Polyethylene oxide (PVA) and polyvinyl alcohol (PVA), which are known to be soluble in water, and the like. The PCL, PDO, PLLA and PLGA polymers may be respectively dissolved in an HFIP organic solvent, and then electrospun can be performed by blending dissolved mussel adhesive protein-Fe ^{3 +} solution as described above. In the case of PEO and PVA polymers, After dissolving each in water, it is possible to perform electrospinning by blending with a solution of mussel adhesive protein-Fe ^{3 +} dissolved in water.

In addition, nanofibers can be prepared by electrospinning by mixing the mussel adhesive protein-Fe ^{3 +} and the biodegradable polymer in various ratios. For example, the biodegradable polymer and the mussel adhesive protein are mixed at a ratio of 90: 10 to 10: 90, preferably 70: 30 or 50: 50 (w / w) can do.

As described above, the mussel adhesive protein can be naturally exposed on the surface of the nanofiber prepared by blending the mussel adhesive protein-Fe ^{3 +} and the biodegradable polymer. In addition, the nanofiber prepared from the mussel adhesive protein-Fe ^{3 +} and the biodegradable polymer show excellent tensile strength and tensile elastic modulus. It has been reported that when a cell is cultured on a support, mechanical stimulation through a support is given, the cell becomes more robust and is very advantageous for tissue regeneration (Kim et al., Nature Biotechnology, 17: 979-983, 1999) . Specifically, in an embodiment of the present invention, an amount of going into the pH of the base of the nanofiber produced according to the present invention Dopa-Fe ^{3 +} complex (Raman spectrum 400-600) becomes a large (FIG. 5), measuring the mechanical properties the pH was confirmed that toward the base that is present DOPA residue is Fe ^{3 +} the number of combining the ions of two, three gradually increase the tensile strength (tensile strength) and tensile modulus (Young's modulus) that in the protein when ( 6 and 7). Thus it can be seen that the mechanical properties increase in proportion linearly as Dopa-Fe ^{3 +} complex is increased.

Furthermore, the behavior of mussel adhesive protein -Fe ^{3 +} and biodegradable after inoculation at a castle nanofibers produced by a non-cyclic polymer bone formation cells such as MC3T3-El cells, 1 hour and 3 days after the cells through the Live / Dead staining image , The number of living cells was observed more than that of nanofibers prepared only with biodegradable polymer (FIG. 8), and more excellent survival ability was confirmed in the cell survival experiment using CCK-8 (FIG. 9).

Therefore, the nanofiber support of the present invention is a biodegradable support having flexibility and elasticity and is expected not only to exert an excellent effect on the regeneration of tissues, but also can easily realize an extracellular matrix, and can be used as a cosmetic, wound dressing or dental matrix And the like.

The present invention using the mussel adhesive protein with the Fe ^{3 +} ions and provide the mechanical properties the increase nanofibers, the nanofibers are various functional physiology using the adhesive force of the mussels coating protein without the need for complex physical and chemical pretreatment on the surface Can be coated with an active material and is very simple in its manufacturing process, is highly industrially applicable, and is excellent in biocompatibility, so that it is suitable as a support for tissue engineering and a medical material.

Figure 1 shows an electron microscope image of nanofibers prepared by blending at various ratios of PCL and mussel adhesive proteins.
Figure 2 shows the results of analysis of DOPA conversion in mussel adhesive proteins through an amino acid analyzer. The peak intensity of DOPA and tyrosine residues indicates a conversion rate of about 37%.
FIG. 3 shows an electron microscope image of nanofibers prepared through various blending with PCL after forming a bond of modified mussel adhesive protein with Fe ^{3 +} .
4 is an image showing changes in color of nanofibers containing mussel adhesive protein and Fe ^{3 +} according to pH.
5 is an image showing raman spectroscopy of a nanofiber containing mussel adhesive protein and Fe ^{3 +} . Purple line means pH 3.0, blue line means pH 5.5 with EDTA, green line means pH 5.5 without EDTA, and red line means Raman spectrum of pH 8.2.
FIG. 6 is a table showing mechanical properties of nanofiber containing mussel adhesive protein and Fe ^{3 +} according to pH. FIG.
FIG. 7 shows strain-stress curves of pH of nanofibers containing mussel adhesive protein and Fe ^{3 +} . FIG.
FIG. 8 is a graph showing the relationship between the nanofibers prepared by mixing PCL-only nanofibers, PCL and fp-1 variant at 90:10 (w / w), and the nanofibers containing mussel adhesive protein and Fe ^{3 +} E1 cells were cultured for toxicity test.
Figure 9 is a nanofiber prepared in the nanofibers, and PCL nanofibers, and mussel adhesive protein -Fe ^{3 +} and biodegradable polymer to fp-1 variant which was prepared by mixing a 90:10 (w / w) produced only PCL . The results of the cell viability test are shown in FIG.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Production of nanofiber based on mussel adhesive protein

1-1. Recombinant mussel adhesive protein fp -One variant Production and production of nanofibers

The mussel adhesive protein fp-1 variant (SEQ ID NO: 3) used in the present invention is a peptide consisting of AKPSYPPTYK (SEQ ID NO: 2) in the amino acid sequence of the mussel adhesive protein fp-1 (Genbank No. Q27409; Was produced in Escherichia coli by the fp-1 mutant that was repeated 12 times. The preparation of the mussel adhesive protein fp-1 variant is the same as that described in International Patent Publication No. WO 2005/092920, which is incorporated herein by reference in its entirety.

Nanofibers were prepared by blending polycaprolactone (PCL) and mussel adhesive protein fp-1 variant solutions to produce nanofibers based on mussel adhesive proteins. Specifically, the mussel adhesive protein fp-1 variant and PCL were dissolved in HFIP (1,1,1,3,3,3-hexafluoroisopropanol) in a concentration of 6 wt%, respectively. The ratio of PCL to fp-1 variant was 90:10 w / w), 70:30 (w / w), and 50:50 (w / w), and electrospinning was performed using this mixed solution. The electrospinning was carried out by passing the mixed solution through a needle having a diameter of 0.4 mm at a rate of 1 ml / h using a syringe pump and generating nanofibers while applying a high voltage of 15 kV, The fibers were placed on an aluminum foil 15 cm away from the needle. As a result, it was confirmed that nanofibers having a straight fiber shape were successfully formed at all the mixing ratios, and these are shown in FIG.

1-2. Recombinant mussel adhesive protein fp -One variant Carry out the correction reaction of

The tyrosinase enzyme (mushroom tyrosinase, SIGMA) was used to modify the tyrosine amino acid in the mussel adhesive protein fp-1 variant prepared in Example 1-1 to DOPA in vitro. Specifically, 150 mg of the lyophilized mussel adhesive protein fp-1 variant and 5 mg of tyrosinase were dissolved in 100 ml of a buffer solution (0.1 M sodium phosphate, 20 mM boric acid, 25 mM Ascorbic acid, pH 6.8) for reaction for 1 hour. After that, dialysis was carried out using 3 L of 1 to 5% acetic acid solution for 3 hours at least for 4 hours, followed by lyophilization. Amino acid composition analysis was performed to analyze the modification efficiency of the mussel adhesive protein fp-1 variant. As a result, it was confirmed that about 37% of all tyrosine residues were converted to DOPA, and the results are shown in FIG.

1-3. Recombinant mussel adhesive protein fp -One variant Wow Fe ^{3 +} Manufacture of nanofibers containing

Since it was confirmed that it is possible to produce nanofibers using the mussel adhesive protein fp-1 through the above <Example 1-1>, experiments for incorporating metal ions into the nanofibers were conducted. Specifically, in Example 1-2, a mussel adhesive protein mfp-1 variant modified with DOPA was dissolved in HFIP at 6 wt%, and a FeCl ₃ solution was mixed so that the molar ratio of DOPA to Fe ^{3 +} was 3: 1 . In this case, Fe ^{3 +} and DOPA are combined and the color of the solution turns dark blue. Electrospinning is performed by blending the solution with PCL solution. As a result, it was confirmed that the nanofibers were successfully produced, and an image obtained using an electron microscope is shown in FIG.

In addition, it was confirmed that the prepared nanofibers changed color depending on the pH. As a result, it was confirmed that the color changes to pink which shows tris bond in pH 8.2 (tris buffer solution) and purple which shows bis bond in pH 5.5 (acetate buffer solution) And treated with EDTA solution to prevent DOPA-metal interactions. These color changes are shown in FIG. 4, respectively.

Example  2. Recombinant mussel adhesive protein fp -One variant Wow Fe ^{3 +} Of nanofiber raman spectroscopy  analysis

Figure 5 shows the analysis of the binding behavior of DOPA and Fe ^{3 +} in nanofibers containing mussel adhesive proteins fp-1 and Fe ^{3 +} using chemically confirmed raman spectroscopy. In FIG. 5, the purple line indicates pH 3.0, the blue line indicates pH 5.5 with EDTA, the green line indicates pH 5.5 without EDTA, and the red line indicates Raman spectrum of pH 8.2. DOPA is combined with the metal containing Fe ^{3 +} is known to the mono-, bis-, tris- combination according to the pH, the Raman spectrum 400 to 600 is in proportion to the amount DOPA-Fe complex on the molar concentration. As shown in FIG. 5, the amount of Dopa-Fe ³⁺ complex increases as the pH increases from 400 to 600 on the Raman spectrum. This indicates that the number of DOPA residues present in the mussel adhesive protein binds to Fe ^{3 +} , And 3, respectively. Also, between 2800 and 3000 on the Raman spectrum shows CH stretching, which is proportional to the molarity of the organic matter present, that is, the molar concentration of nanofibers. Therefore, [Raman 400 ~ 600 intensity] / [Raman 2800-3200 intensity] indicates the amount of DOPA-Fe complex per mole of nanofibers, and the amount of DOPA-Fe complex per mole of nanofibers increases with increasing pH.

Example  3. Recombinant mussel adhesive protein fp -One variant Wow Fe ^{3 +} Analysis of Mechanical Properties of Nanofibers Contained

To investigate the mechanical properties of the nanofibers containing the recombinant mussel adhesion proteins fp-1 and Fe ^{3 +} , the nanofibers prepared in Example 1-3 were dissolved in a pH 8.2 solution, a pH 5.5 solution and an EDTA solution, respectively After dipping, each of the nanofibers was cut into 10 mm x 25 mm size without drying, and then pulled at a rate of 5 mm / min using a universal testing machine (INSTRON) 2 kN load cell.

FIG. 6 shows the mechanical properties of nanofibers containing mussel adhesive protein and Fe ^{3 +} according to pH, and FIG. 7 shows strain-stress curves according to pH of nanofibers containing mussel adhesive protein and Fe ^{3 +} . The tensile strength and Young's modulus of EDTA, pH 5.5, and pH 8.2 were gradually increased in the order of pH and pH, Tensile strength and tensile elastic modulus were the maximum values in pH 8.2 solution. Therefore, it can be seen that the pH of the mechanical properties, as increasingly ^{Dopa-Fe 3 + complex (Raman} 400 ~ 600) is increased to a base proportional increase linearly.

Example  4. Recombinant mussel adhesive protein fp -One variant Wow Fe ^{3 +} Analysis of Cellular Experiments of Nanofibers Containing

It is possible to easily realize the extracellular matrix using the supporter of the present invention including the recombinant mussel adhesion protein fp-1 and Fe ^{3 +} , and it can be utilized as a medical material such as cosmetics, wound covering materials and dental matrix , Cell experiments were performed using MC3T3-El cells, which are non-transformed osteogenic cells. The nanofibers used in the cell experiments were prepared by dissolving the mussel adhesive protein fp-1 variant and PCL in 6 wt% of HFIP (1,1,1,3,3,3-hexafluoroisopropanol), respectively. The ratio of PCL to fp-1 variant 90:10 (w / w), followed by electrospinning using this mixed solution. Then, the nanofibers containing Fe ^{3 +} were produced by the method described in the above <Example 1-3>.

The biodegradable polymer is PCL only made of nanofibers, and fp PCL-1 variant a ratio of 90:10 (w / w) to the mixture gave nanofibers, and Fe ^{+ 3,} respectively MC3T3-El cells at nanofibers containing the dose of After 1, 3, and 3 days, we observed the behavior of cells through Live / Dead staining image. As a result, more viable cells were observed in nanofibers and Fe ^{3 +} -containing nanofibers mixed with PCL and fp-1 variants at a ratio of 90:10 (w / w), compared with nanofibers made only with PCL Green represents living cells, red represents dead cells). The cell viability was measured using a CCK-8 (Dojindo) reagent. As a result, the nanofibers mixed with PCL and fp-1 variants at a ratio of 90:10 (w / w) We could observe more survival of the cells after 3 days in Fe ^{3 +} containing nanofibers. Therefore, through the cell experiments of MC3T3-El cells, osteoblasts, the supports of the present invention were confirmed to be applicable to various medical materials.

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Electrospinning nanofibers reinforced by mussel coating protein          and their application <130> DPP20134401KR <160> 26 <170> Kopatentin 1.71 <210> 1 <211> 565 <212> PRT <213> Artificial Sequence <220> <223> fp-1 <400> 1 Met Glu Gly Ile Lys Leu Asn Leu Cys Leu Leu Cys Ile Phe Thr Phe   1 5 10 15 Asp Val Leu Gly Phe Ser Asn Gly Asn Ile Tyr Asn Ala His Val Ser              20 25 30 Ser Tyr Ala Gly Ala Ser Ala Gly Ala Tyr Lys Lys Leu Pro Asn Ala          35 40 45 Tyr Pro Tyr Gly Thr Lys Pro Glu Pro Val Tyr Lys Pro Val Lys Thr      50 55 60 Ser Tyr Ser Ala Pro Tyr Lys Pro Pro Thr Tyr Gln Gln Leu Lys Lys  65 70 75 80 Lys Val Asp Tyr Arg Pro Thr Lys Ser Tyr Pro Pro Thr Tyr Gly Ser                  85 90 95 Lys Thr Asn Tyr Leu Pro Leu Ala Lys Lys Leu Ser Ser Tyr Lys Pro             100 105 110 Ile Lys Thr Thr Tyr Asn Ala Lys Thr Asn Tyr Pro Pro Val Tyr Lys         115 120 125 Pro Lys Met Thr Tyr Pro Pro Thr Tyr Lys Pro Lys Pro Ser Tyr Pro     130 135 140 Pro Thr Tyr Lys Ser Lys Pro Thr Tyr Lys Pro Lys Ile Thr Cys Pro 145 150 155 160 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Lys                 165 170 175 Lys Thr Tyr Pro Pro Thr Tyr Lys Pro Lys Val Thr Tyr Pro Pro Thr             180 185 190 Tyr Lys Pro Lys Pro Ser Tyr Pro Pro Ile Tyr Lys Ser Lys Pro Thr         195 200 205 Tyr Lys Pro Lys Ile Thr Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser     210 215 220 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 225 230 235 240 Ala Lys Pro Thr Tyr Lys Ala Lys Pro Thr Tyr Pro Ser Thr Tyr Lys                 245 250 255 Ala Lys Pro Thr Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro             260 265 270 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys         275 280 285 Pro Thr Tyr Ile Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys     290 295 300 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 305 310 315 320 Tyr Lys Ala Lys Ser Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Thr                 325 330 335 Tyr Lys Ala Lys Pro Thr Tyr Pro Ser Thr Tyr Lys Ala Lys Pro Ser             340 345 350 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Thr Tyr Lys Ala Lys Pro Thr         355 360 365 Tyr Pro Ser Thr Tyr Lys Ala Lys Pro Thr Tyr Pro Ser Thr Tyr Lys     370 375 380 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Lys Ile Ser Tyr Pro 385 390 395 400 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Ser Thr Tyr Lys Ala Lys                 405 410 415 Ser Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Ser Ser Tyr Pro Pro Thr             420 425 430 Tyr Lys Ala Lys Pro Thr Tyr Lys Ala Lys Pro Thr Tyr Pro Ser Thr         435 440 445 Tyr Lys Ala Lys Pro Thr Tyr Lys Ala Lys Pro Thr Tyr Pro Pro Thr     450 455 460 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Lys Pro Ser 465 470 475 480 Tyr Pro Pro Thr Tyr Lys Ser Lys Ser Ser Tyr Pro Ser Ser Tyr Lys                 485 490 495 Pro Lys Lys Thr Tyr Pro Pro Thr Tyr Lys Pro Lys Leu Thr Tyr Pro             500 505 510 Pro Thr Tyr Lys Pro Lys Pro Ser Tyr Pro Pro Ser Tyr Lys Pro Lys         515 520 525 Ile Thr Tyr Pro Ser Thr Tyr Lys Leu Lys Pro Ser Tyr Pro Pro Thr     530 535 540 Tyr Lys Ser Lys Thr Ser Tyr Pro Pro Thr Tyr Asn Lys Lys Ile Ser 545 550 555 560 Tyr Pro Ser Gln Tyr                 565 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> Fragment sequence derived from fp-1 <400> 2 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys   1 5 10 <210> 3 <211> 16 <212> PRT <213> Artificial Sequence <220> Fragment sequence derived from fp-1-RGD <400> 3 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Gly Arg Gly Asp Ser Pro   1 5 10 15 <210> 4 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> fp-1 variant <400> 4 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro   1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys              20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr          35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser      50 55 60 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys  65 70 75 80 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro                  85 90 95 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys             100 105 110 Pro Ser Tyr Pro Pro Thr Tyr Lys         115 120 <210> 5 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> fp-1 variant-RGD <400> 5 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro   1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys              20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr          35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser      50 55 60 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys  65 70 75 80 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro                  85 90 95 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys             100 105 110 Pro Ser Tyr Pro Pro Thr Tyr Lys Gly Arg Gly Asp Ser Pro         115 120 125 <210> 6 <211> 456 <212> PRT <213> Artificial Sequence <220> <223> fp-2 <400> 6 Thr Asn Arg Pro Asp Tyr Asn Asp Asp Glu Glu Asp Asp Tyr Lys Pro   1 5 10 15 Pro Val Tyr Lys Pro Ser Ser Ser Ser Tyr Arg Pro Val Asn Pro Cys              20 25 30 Leu Lys Lys Pro Cys Lys Tyr Asn Gly Val Cys Lys Pro Arg Gly Gly          35 40 45 Ser Tyr Lys Cys Phe Cys Lys Gly Gly Tyr Tyr Gly Tyr Asn Cys Asn      50 55 60 Leu Lys Asn Ala Cys Lys Pro Asn Gln Cys Lys Asn Lys Ser Arg Cys  65 70 75 80 Val Pro Val Gly Lys Thr Phe Lys Cys Val Cys Arg Asn Gly Asn Phe                  85 90 95 Gly Arg Leu Cys Glu Lys Asn Val Cys Ser Pro Asn Pro Cys Lys Asn             100 105 110 Asn Gly Lys Cys Ser Pro Leu Gly Lys Thr Gly Tyr Lys Cys Thr Cys         115 120 125 Ser Gly Gly Tyr Thr Gly Pro Arg Cys Glu Val His Ala Cys Lys Pro     130 135 140 Asn Pro Cys Lys Asn Lys Gly Arg Cys Phe Pro Asp Gly Lys Thr Gly 145 150 155 160 Tyr Lys Cys Arg Cys Val Asp Gly Tyr Ser Gly Pro Thr Cys Gln Glu                 165 170 175 Asn Ala Cys Lys Pro Asn Pro Cys Ser Asn Gly Gly Thr Cys Ser Ala             180 185 190 Asp Lys Phe Gly Asp Tyr Ser Cys Glu Cys Arg Pro Gly Tyr Phe Gly         195 200 205 Pro Glu Cys Glu Arg Tyr Val Cys Ala Pro Asn Pro Cys Lys Asn Gly     210 215 220 Gly Ile Cys Ser Ser Asp Gly Ser Gly Gly Tyr Arg Cys Arg Cys Lys 225 230 235 240 Gly Gly Tyr Ser Gly Pro Thr Cys Lys Val Asn Val Cys Lys Pro Thr                 245 250 255 Pro Cys Lys Asn Ser Gly Arg Cys Val Asn Lys Gly Ser Ser Tyr Asn             260 265 270 Cys Ile Cys Lys Gly Gly Tyr Ser Gly Pro Thr Cys Gly Glu Asn Val         275 280 285 Cys Lys Pro Asn Pro Cys Gln Asn Arg Gly Arg Cys Tyr Pro Asp Asn     290 295 300 Ser Asp Asp Gly Phe Lys Cys Arg Cys Val Gly Gly Tyr Lys Gly Pro 305 310 315 320 Thr Cys Glu Asp Lys Pro Asn Pro Cys Asn Thr Lys Pro Cys Lys Asn                 325 330 335 Gly Gly Lys Cys Asn Tyr Asn Gly Lys Ile Tyr Thr Cys Lys Cys Ala             340 345 350 Tyr Gly Trp Arg Gly Arg His Cys Thr Asp Lys Ala Tyr Lys Pro Asn         355 360 365 Pro Cys Val Val Ser Lys Pro Cys Lys Asn Arg Gly Lys Cys Ile Trp     370 375 380 Asn Gly Lys Ala Tyr Arg Cys Lys Cys Ala Tyr Gly Tyr Gly Gly Arg 385 390 395 400 His Cys Thr Lys Lys Ser Tyr Lys Lys Asn Pro Cys Ala Ser Arg Pro                 405 410 415 Cys Lys Asn Arg Gly Lys Cys Thr Asp Lys Gly Asn Gly Tyr Val Cys             420 425 430 Lys Cys Ala Arg Gly Tyr Ser Gly Arg Tyr Cys Ser Leu Lys Ser Pro         435 440 445 Pro Ser Tyr Asp Asp Asp Glu Tyr     450 455 <210> 7 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> fp-3 <400> 7 Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly   1 5 10 15 Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp              20 25 30 Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr          35 40 45 <210> 8 <211> 787 <212> PRT <213> Artificial Sequence <220> <223> fp-4 <400> 8 Tyr Gly Arg Arg Tyr Gly Glu Pro Ser Gly Tyr Ala Asn Ile Gly His   1 5 10 15 Arg Arg Tyr Tyr Glu Arg Ala Ser Ser Phe His Arg His His Val              20 25 30 His Gly His His Leu Leu His Arg His Val His Arg His Ser Val Leu          35 40 45 His Gly His Val His Met His Arg Val Ser His Arg Ile Met His Arg      50 55 60 His Arg Val Leu His Gly His Val His Arg His Arg Val Leu His Arg  65 70 75 80 His Val His Arg Arg His Val Leu His Gly His Val His Arg Arg His Arg                  85 90 95 Val Leu His Arg His Leu His Arg His Arg Val Leu His Gly His Val             100 105 110 His Arg His Arg Val Leu His Asn His Val His Arg His Ser Val Leu         115 120 125 His Gly His Val His Arg His Arg Val Leu His Arg His Val Val His Arg     130 135 140 His Asn Val Leu His Gly His Val His Arg Arg His Val Leu His Lys 145 150 155 160 His Val His Asp His Arg Val Leu His Lys His Leu His Lys His Gln                 165 170 175 Val Leu His Gly His Val His Arg His Gln Val Leu His Lys His Val             180 185 190 His Asn His Arg Val Leu His Lys His Leu His Lys His Gln Val Leu         195 200 205 His Gly His Val His Thr His Arg Val Leu His Lys His Val His Lys     210 215 220 His Arg Val Leu His Lys His Leu His Lys His Gln Val Leu His Gly 225 230 235 240 His Ile His Thr His Arg Val Leu His Lys His Leu His Lys His Gln                 245 250 255 Val Leu His Gly His Val His Thr His Arg Val Leu His Lys His Val             260 265 270 His Lys His Arg Val Leu His Lys His Leu His Lys His Gln Val Leu         275 280 285 His Gly His Val His Met His Arg Val Leu His Lys His Val His Lys     290 295 300 His Arg Val Leu His Lys His Val His Lys His His Val Val His Lys 305 310 315 320 His Val His Ser His Arg Val Leu His Lys His Val His Lys His Arg                 325 330 335 Val Glu His Gln His Val His Lys His His Val Leu His Arg His Val             340 345 350 His Ser His His Val Val His Ser Val Val His Lys His Arg Val Val         355 360 365 His Ser His Val His Lys His Asn Val Val His Ser His Val His Arg     370 375 380 His Gln Ile Leu His Arg His Val His Arg His Gln Val Val His Arg 385 390 395 400 His Val His Arg His Leu Ile Ala His Arg His Ile His Ser His Gln                 405 410 415 Ala Ala Val His Arg His Val His Thr His Val Phe Glu Gly Asn Phe             420 425 430 Asn Asp Asp Gly Thr Asp Val Asn Leu Arg Ile Arg His Gly Ile Ile         435 440 445 Tyr Gly Gly Asn Thr Tyr Arg Leu Ser Gly Gly Arg Arg Arg Phe Met     450 455 460 Thr Leu Trp Gln Glu Cys Leu Glu Ser Tyr Gly Asp Ser Asp Glu Cys 465 470 475 480 Phe Val Gln Leu Gly Asn Gln His Leu Phe Thr Val Val Gln Gly His                 485 490 495 His Ser Thr Ser Phe Arg Ser Asp Leu Ser Asn Asp Leu His Pro Asp             500 505 510 Asn Asn Ile Glu Gln Ile Ala Asn Asp His Val Asn Asp Ile Ala Gln         515 520 525 Ser Thr Asp Gly Asp Ile Asn Asp Phe Ala Asp Thr His Tyr Asn Asp     530 535 540 Val Ala Pro Ile Ala Asp Val His Val Asp Asn Ile Ala Gln Thr Ala 545 550 555 560 Asp Asn His Val Lys Asn Ile Ala Gln Thr Ala His His His Val Asn                 565 570 575 Asp Val Ala Gln Ile Ala Asp Asp His Val Asn Asp Ile Gly Gln Thr             580 585 590 Ala Tyr Asp His Val Asn Asn Ile Gly Gln Thr Ala Asp Asp His Val         595 600 605 Asn Asp Ile Ala Gln Thr Ala Asp Asp His Val Asn Ale Ile Ala Gln     610 615 620 Thr Ala Asp Asp His Val Asn Ala Ile Ala Gln Thr Ala Asp His Val 625 630 635 640 Asn Asp Ile Gly Asp Thr Ala Asn Ser Ile Val Val Arg Gl Gln Gly                 645 650 655 Val Ala Lys Asn His Leu Tyr Gly Ile Asn Lys Ala Ile Gly Lys His             660 665 670 Ile Gln His Leu Lys Asp Val Ser Asn Arg His Ile Glu Lys Leu Asn         675 680 685 Asn His Ala Thr Lys Asn Leu Leu Gln Ser Ala Leu Gln His Lys Gln     690 695 700 Gln Thr Ile Glu Arg Glu Ile Gln His Lys Arg His Leu Ser Glu Lys 705 710 715 720 Glu Asp Ile Asn Leu Gln His Glu Asn Ala Met Lys Ser Lys Val Ser                 725 730 735 Tyr Asp Gly Pro Val Phe Asn Glu Lys Val Ser Val Val Ser Asn Gln             740 745 750 Gly Ser Tyr Asn Gly Lys Val Pro Val Leu Ser Asn Gly Gly Gly Tyr         755 760 765 Asn Gly Lys Val Ser Ala Leu Ser Asp Gln Gly Ser Tyr Asn Glu Gly     770 775 780 Tyr Ala Tyr 785 <210> 9 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> fp-5 <400> 9 Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His   1 5 10 15 Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr              20 25 30 Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys          35 40 45 Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg      50 55 60 Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser  65 70 75 <210> 10 <211> 99 <212> PRT <213> Artificial Sequence <220> <223> fp-6 <400> 10 Gly Gly Asn Tyr Arg Gly Tyr Cys Ser Asn Lys Gly Cys Arg Ser   1 5 10 15 Gly Tyr Ile Phe Tyr Asp Asn Arg Gly Phe Cys Lys Tyr Gly Ser Ser              20 25 30 Ser Tyr Lys Tyr Asp Cys Gly Asn Tyr Ala Gly Cys Cys Leu Pro Arg          35 40 45 Asn Pro Tyr Gly Arg Val Lys Tyr Tyr Cys Thr Lys Lys Tyr Ser Cys      50 55 60 Pro Asp Phe Tyr Tyr Tyr Asn Asn Lys Gly Tyr Tyr Tyr Tyr Asn  65 70 75 80 Asp Lys Asp Tyr Phe Asn Cys Gly Ser Tyr Asn Gly Cys Cys Leu Arg                  85 90 95 Ser Gly Tyr             <210> 11 <211> 196 <212> PRT <213> Artificial Sequence <220> <223> fp-151 <400> 11 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr   1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala              20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro          35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu      50 55 60 Gly Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser  65 70 75 80 Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys                  85 90 95 Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly             100 105 110 Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr         115 120 125 Lys Lys Tyr Tyr Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr     130 135 140 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 145 150 155 160 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys                 165 170 175 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro             180 185 190 Pro Thr Tyr Lys         195 <210> 12 <211> 202 <212> PRT <213> Artificial Sequence <220> <223> fp-151-RGD <400> 12 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr   1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala              20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro          35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu      50 55 60 Gly Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser  65 70 75 80 Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys                  85 90 95 Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly             100 105 110 Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr         115 120 125 Lys Lys Tyr Tyr Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr     130 135 140 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 145 150 155 160 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys                 165 170 175 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro             180 185 190 Pro Thr Tyr Lys Gly Arg Gly Asp Ser Pro         195 200 <210> 13 <211> 172 <212> PRT <213> Artificial Sequence <220> <223> fp-131 <400> 13 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr   1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala              20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro          35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Trp Ala      50 55 60 Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly  65 70 75 80 Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn                  85 90 95 Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly Ser Ala             100 105 110 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro         115 120 125 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro     130 135 140 Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr 145 150 155 160 Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Leu                 165 170 <210> 14 <211> 175 <212> PRT <213> Artificial Sequence <220> <223> fp-353 <400> 14 Met Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly   1 5 10 15 Gly Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly              20 25 30 Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Glu          35 40 45 Phe Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Ser Asn      50 55 60 His Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly  65 70 75 80 Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr                  85 90 95 Lys Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His             100 105 110 Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser Lys Leu Ala         115 120 125 Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly     130 135 140 Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn 145 150 155 160 Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Leu Glu                 165 170 175 <210> 15 <211> 189 <212> PRT <213> Artificial Sequence <220> <223> fp-153 <400> 15 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr   1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala              20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro          35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Glu Phe Ser      50 55 60 Ser Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Ser Asn His Tyr  65 70 75 80 His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys                  85 90 95 Gly Lys Tyr Tyr Gys Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn             100 105 110 Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys         115 120 125 Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser Lys Leu Ala Asp Tyr     130 135 140 Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly Asn Tyr 145 150 155 160 Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn Asn Gly                 165 170 175 Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Leu Glu             180 185 <210> 16 <211> 189 <212> PRT <213> Artificial Sequence <220> <223> fp-351 <400> 16 Met Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly   1 5 10 15 Gly Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly              20 25 30 Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Glu          35 40 45 Phe Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Ser Asn      50 55 60 His Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly  65 70 75 80 Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr                  85 90 95 Lys Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His             100 105 110 Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser Lys Leu Ala         115 120 125 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro     130 135 140 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro 145 150 155 160 Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr                 165 170 175 Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Leu Glu             180 185 <210> 17 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 1 <400> 17 Arg Gly Asp   One <210> 18 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 2 <400> 18 Arg Gly Asp Ser   One <210> 19 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 3 <400> 19 Arg Gly Asp Cys   One <210> 20 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 4 <400> 20 Arg Gly Asp Val   One <210> 21 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 5 <400> 21 Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro   1 5 10 <210> 22 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 6 <400> 22 Gly Arg Gly Asp Ser   1 5 <210> 23 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 7 <400> 23 Gly Arg Gly Asp Thr Pro   1 5 <210> 24 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 8 <400> 24 Gly Arg Gly Asp Ser Pro   1 5 <210> 25 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 9 <400> 25 Gly Arg Gly Asp Ser Pro Cys   1 5 <210> 26 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 10 <400> 26 Tyr Arg Gly Asp Ser   1 5

Claims (12)

A support for nanofiber tissue engineering comprising a complex of 3,4-dihydroxyphenyl-L-alanine (dopa) bound to a mussel adhesive protein and Fe ³⁺ ions and a biodegradable polymer, Wherein the protein is at least 20% of the total number of tyrosine residues contained therein is a waveguide-
Depending on pH conditions, color and intensity change,
Support for nanofiber tissue engineering.
The support for nanofiber tissue engineering according to claim 1, wherein the surface of the nanofibers is coated with a physiologically active substance such as a cell, a protein, a nucleic acid, a sugar or an enzyme.
The biodegradable polymer according to claim 1, wherein the biodegradable polymer is selected from the group consisting of polycaprolactone (PCL), polydioxanone (PDO), poly (L-lactide), PLGA (poly- Or polyvinyl alcohol (PVA).
The mussel adhesive protein according to claim 1, wherein the mussel adhesive protein comprises a protein consisting of the amino acid sequence of SEQ ID NO: 1, a protein having an amino acid sequence of SEQ ID NO: 2 connected continuously one to 12 times, A protein consisting of the amino acid sequence of SEQ ID NO: 8, a protein consisting of the amino acid sequence of SEQ ID NO: 9, and a protein consisting of the amino acid sequence of SEQ ID NO: 10, Wherein said protein is a fusion protein linked to said protein.
5. The nanofiber tissue engineering support according to claim 4, wherein the protein in which the amino acid sequence of SEQ ID NO: 2 is connected in a series of 12 to 12 consecutive amino acids is a protein consisting of the amino acid sequence of SEQ ID NO:
The fusion protein according to claim 4, wherein the fusion protein comprises a fusion protein comprising the amino acid sequence of SEQ ID NO: 11, a fusion protein comprising the amino acid sequence of SEQ ID NO: 13, a fusion protein comprising the amino acid sequence of SEQ ID NO: And a fusion protein consisting of the amino acid sequence of SEQ ID NO: 16.
2. The support for nanofiber tissue engineering according to claim 1, wherein the mussel adhesive protein is a polypeptide chain comprising 3 to 25 amino acids including RGD (Arg Gly Asp) at the carboxyl terminal or amino terminal thereof.
8. The nanofiber tissue engineering support according to claim 7, wherein the polypeptide comprising RGD is an amino acid sequence selected from the group consisting of SEQ ID NO: 17 to SEQ ID NO: 26.
9. The nanofiber tissue engineering support according to claim 8, wherein the mussel adhesive protein is a protein consisting of the amino acid sequence of SEQ ID NO: 3, a protein consisting of the amino acid sequence of SEQ ID NO: 5, or a protein consisting of the amino acid sequence of SEQ ID NO:
[3] The method of claim 1, wherein the ratio of the biodegradable polymer and the conjugate of 3,4-dihydroxyphenyl-L-alanine (dopa) and Fe ³⁺ bonded to the mussel adhesive protein is 90 to 50: 50 (w / w). &Lt; / RTI &gt;
(a) adding to a solution having a pH of 11 or more containing a mussel adhesive protein in which 20% or more of the total number of tyrosine residues contained in the mussel adhesive protein is modified with 3,4-dihydroxyphenyl-L-alanine step by the addition of a reagent that provides ³⁺ ions to combine the wave director and the Fe ³⁺ ions in the mussel adhesive protein; And
(b) preparing a spinning solution by adding a biodegradable polymer and electrospinning the spinning solution,
A method of making the nanofiber tissue engineering support of claim 1.
The biodegradable polymer according to claim 11, wherein the biodegradable polymer is selected from the group consisting of polycaprolactone (PCL), polydioxanone (PDO), poly (L-lactide), PLGA (poly- Or PVA (polyvinyl alcohol).

Images