KR101578522B1 - Mussel adhesive protein-based bioadhesive hydrogel - Google Patents

Abstract

The present invention relates to a bioadhesive hydrogel based on mussel adhesive protein and a process for its preparation.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a mussel adhesive protein-based bioadhesive hydrogel,

The present invention relates to a bioadhesive hydrogel based on mussel adhesive protein and a process for its preparation.

A bioadhesive refers to a substance having adhesion properties to various biological samples such as biological membranes, cell walls, lipids, proteins, DNA, growth factors, cells and tissues such as proteins, DNA, growth factors and cells. A variety of biomedical applications are possible, including engineering supports, drug delivery carriers, tissue fillers, wound healing, or intestinal adhesion prevention. Biodegradable adhesives require strong adhesion and crosslinking capabilities and should retain their function in the organism for a long time. Examples of the biocompatible adhesives currently commercialized or put to practical use include cyanoacrylate instant adhesive, fibrin glue, gelatin glue and polyurethane adhesive. However, in the case of a bioadhesive using a synthetic polymer, a very weak force is exhibited in the presence of an aqueous solution in a living body, and cyanoacrylate-based bioadhesive has been pointed out as a serious limitation to cause side effects such as immune reaction to the human body. In addition, in the case of the fibrin-based bioadhesive which is currently used in actual patients, the side effect is small, but its adhesive ability is very low, which limits its use. In the case of a gelatinous tissue adhesive, formalin or glutaraldehyde used as a crosslinking agent also undergoes a cross-linking reaction with a protein in the living body to cause tissue toxicity, and a polyurethane-based tissue adhesive has a problem that an aromatic diisocyanate as a starting material for synthesis is biocompatible .

Therefore, in order to overcome such a problem, it is necessary to develop an ideal form of biomaterial having a strong adhesion and crosslinking ability on the wet tissue surface and showing minimal side effects in vivo.

On the other hand, marine life mussel produces and secretes adhesive proteins, which can firmly attach the mussel itself to wet solid surfaces such as rocks in the sea, thereby affecting the impact of waves and the buoyancy effect of seawater. I do not accept. Mussel adhesive protein is known as a strong natural adhesive. It has flexibility that can be flexed while showing high tensile strength about twice that of epoxy resin, compared with chemical synthetic adhesive. In addition, mussel adhesive proteins have the ability to adhere to a variety of surfaces, such as plastics, glass, metals, teflon and biomaterials. Adhesion on wet surfaces, which remain an incomplete challenge in chemical adhesive development, It is possible within.

In particular, in the case of US-blocks scan edul-less foot protein 1 (Mefp-1) proteins that make up the byssus of the mussel (byssal thread), a dihydroxy-phenylalanine (DOPA) residues present in the protein, while in combination with Fe ^{3 +} ion surface And 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. However, there has been no attempt to make a hydrogel or apply it as a bioadhesive using the binding of DOPA to metal and the oxidation reaction of DOPA in mussel adhesive proteins.

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 a mass production technology, the present inventors have converted a tyrosine residue of a mussel adhesive protein mass-produced in an Escherichia coli system to DOPA with high efficiency, and used it to prepare a hydrogel based on a mussel adhesive protein. The present inventors have confirmed that the gel exhibits excellent efficacy as a bioadhesive agent, thereby completing the present invention.

It is an object of the present invention to provide a hydrogel containing a mussel adhesive protein and a method for producing the same, and further to provide a bioadhesive comprising the mussel adhesive protein-based hydrogel.

Specifically, one object of the present invention is to provide a bioadhesive hydrogel comprising a mussel adhesive protein.

It is still another object of the present invention to provide a method for producing the bioadhesive hydrogel.

It is another object of the present invention to provide a bioadhesive composition comprising the hydrogel.

It is another object of the present invention to provide a tissue engineering support comprising the hydrogel.

Another object of the present invention is to provide a drug delivery carrier comprising the hydrogel.

In one aspect, the present invention relates to a hydrogel comprising a mussel adhesive protein and a method of making the same.

In the present invention, the term "hydrogel " means a material in which a large amount of water is filled in a lattice of a polymer material and swelled to maintain a three-dimensional structure, and has a liquid-like shape. Hydrogels can absorb at least 20% of the total weight of water and, after the hydrogel is swollen in solution, are thermodynamically stable and have mechanical and physicochemical properties corresponding to the intermediate form of liquid and solid.

In the present invention, "mussel adhesive protein" is a mussel-derived adhesive protein, preferably Mytilus edulis), the US's tiller ropes Robin's going to Borealis City (Mytilus galloprovincialis), or US tiller nose Ruth Curse (Mytilus but are not limited to, mussel adhesive proteins or variants thereof derived from coruscus .

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 A protein selected from the group consisting of fp-3 (SEQ ID NO: 5), fp-3 (SEQ ID NO: 6), fp-4 (SEQ ID NO: 7), fp-5 (SEQ ID NO: 8), 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, 15). ≪ / 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 fp-1 variant polypeptide (SEQ ID NO: 3) that is connected in 12 consecutive sequences.

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: 16), RGDS (Arg Gly Asp Ser, SEQ ID NO: 17), RGDC (Arg Gly Asp Cys, SEQ ID NO: 18), RGDV (Arg Gly Asp Val, SEQ ID NO: 19), RGDSPASSKP (Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro, SEQ ID NO: 20), GRGDS (Gly Arg Gly Asp Ser, SEQ ID NO: (SEQ ID NO: 23), GRGDSP (Gly Arg Gly Asp Ser Pro Cys, SEQ ID NO: 24) and YRGDS (Tyr Arg Gly Asp Ser, SEQ ID NO: No. 25) 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: 4 1 variant-RGD polypeptide consisting of the amino acid sequence of SEQ ID NO: 11, or an fp-151-RGD polypeptide consisting of the amino acid sequence of SEQ ID NO: 11.

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 meets the DOPA conversion and solubility conditions through a modification reaction with tyrosinase in vitro (FIG. 1).

The present invention provides a bioadhesive hydrogel based on mussel adhesive protein.

Preferably, the hydrogel based on the mussel adhesive protein in the present invention may be a hydrogel formed by binding of DOPA residues and Fe ^{3 +} ions contained in the mussel adhesive protein.

Accordingly, in a preferred embodiment, the present invention provides a method for producing a bioadhesive hydrogel comprising reacting a mussel adhesive protein and Fe ^& lt ^{; 3 + &} gt ^; ions.

The method comprises preparing a hydrogel by combining DOPA and Fe ^{3 +} ions contained in a 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 hydrogel can be formed by raising the pH to 11 or more by using NaOH.

Also preferably, the hydrogel may be formed by oxidation of the DOPA residue contained in the mussel adhesive protein in the present invention.

Accordingly, in another preferred embodiment, the present invention provides a method for producing a bioadhesive hydrogel comprising oxidizing a DOPA residue contained in a mussel adhesive protein.

The method is a step of preparing a hydrogel by oxidation of DOPA contained in a mussel adhesive protein. When DOPA is oxidized, the DOPA quinone is converted into the DOPA-DOPA bond, or the DOPA quinone bonds with the surrounding amine group or thiol group.

In the above method, NaIO ₄ is preferably used to oxidize DOPA contained in the mussel adhesive protein, but not limited thereto. More preferably, the NaIO ₄ solution is added so that the ratio of DOPA: IO ₄ ^- molecules is preferably less than 2: 1 or IO ₄ ^- to the solution in which the mussel adhesive protein is dissolved in 30 to 50 wt% Hydrogels can be formed.

In a specific example of the present invention, a photograph of the hydrogel formed by the above two methods is shown in FIG. 2, and it is confirmed that the color change by the pH change and the oxidation is made according to the conventionally known reports, Respectively. The binding pattern of DOPA and Fe ^{3 +} was chemically analyzed and shown in FIG.

In addition, the rheological properties of hydrogels prepared by the above two methods were analyzed. Through the rheological analysis, the storage modulus, loss modulus, and loss tangent delta value according to time, frequency, and strain are calculated. And it is possible to analyze gel formation time, mechanical strength, and self-resilience. As a rheological analysis method, a rheological analyzer in which a parallel plate having a diameter of 25 mm is rotated is used in the present invention, but the present invention is not limited thereto. Each sample was added immediately after the gel reaction was initiated and preferably the same amount was added to cover a portion of the plate, but not more than that. As a result of analyzing the rheological properties of the hydrogel prepared by the above-mentioned two methods, it was found that both of the cases had the property of being gelated (FIGS. 3 to 6).

The hydrogel has mechanical flexibility similar to that of the actual tissue, and it contains a lot of water, but since the bond of the gel is not cut off by the water, it is necessary to adhere to the wet surface including moisture and resist to the external moisture It has been actively applied to a medical adhesive to be used. Therefore, the hydrogel having excellent tissue adhesiveness according to the present invention can be applied to various biomedical applications such as a tissue adhesive or a hemostatic agent, a tissue engineering support, a drug delivery carrier, a tissue filler, wound healing, or prevention of intestinal adhesion.

Accordingly, in another aspect, the present invention relates to a bioadhesive composition comprising a hydrogel based on mussel adhesive protein.

Specifically, in the present invention, the possibility of a tissue adhesive, which is one of uses of a hydrogel containing a mussel adhesive protein, was examined. Specifically, in the examples, when the mussel adhesive protein hydrogels formed by the above two methods were put in between porcine skin grafts, respectively, and reacted for 2 hours in water, (50 to 200 KPa) (Fig. 11 and Fig. 12). Further, it was confirmed through continuous adhesion measurement that the hydrogel containing the mussel adhesive protein and Fe ^{3 +} had self-resilience (Fig. 13).

The bioadhesive composition of the present invention can be used in various fields such as skin, blood vessels, digestive system, cranial nerve, plastic surgery, orthopedic surgery, etc. instead of cyanoacrylic adhesive or fibrin adhesive which are currently used in the market. For example, the biocompatible biodegradable adhesive of the present invention can replace surgical sutures, can be used to block unnecessary blood vessels, and can be used for soft tissue such as facial tissue, cartilage, and hard tissue hemostasis and suture such as bones and teeth And it is possible to apply it as a home remedy. Various application fields of the biocompatible bioadhesive composition of the present invention are summarized as follows:

In one embodiment, the bioadhesive of the present invention can be applied to the internal and external surfaces of the human body. That is, the bioadhesive of the present invention can be applied to the external surface of the human body such as skin, Lt; / RTI > In addition, the bioadhesive of the present invention can be used to adhere damaged portions of tissue or seal air / fluid leaks in tissue, to adhere medical devices to tissues, or to fill defective portions of tissue. As used herein, the term "living tissue" is not particularly limited and includes, for example, skin, bone, nerve, axon, cartilage, blood vessel, cornea, muscle, fascia, brain, prostate, breast, endometrium, , Liver, testis, ovary, cervix, rectum, stomach, lymph node, bone marrow, and kidney.

In another embodiment, the bioadhesive of the present invention can be used for wound healing. For example, the biocompatible bioadhesive of the present invention can be used as a dressing applied to a wound.

In another embodiment, the bioadhesive of the present invention can be used for skin suture. That is, the bioadhesive of the present invention can be applied locally to seal the wound, thereby replacing the suture. In addition, the bioadhesive of the present invention can be applied to the hernia repair, for example, for surface coating of meshes used for hernia repair.

In another embodiment, the bioadhesive of the present invention can also be used to prevent suture and leakage of tubing such as blood vessels. Further, the bioadhesive of the present invention can be used for hemostasis.

In another embodiment, the bioadhesive of the present invention can be used as an anti-adhesion agent after surgery. Adhesion occurs at all surgical sites and is a phenomenon where other tissues stick around the wound around the surgical site. Adhesion occurs in about 97% after surgery, and in particular, 5-7% of them cause serious problems. In order to prevent such adhesion, wound minimization or anti-inflammatory drugs may be used. In addition, TPA (tissue plasminogen activator) is activated or physical barriers such as crystalline solution, polymer solution, and solid membrane are used to prevent the formation of fibrin. However, these methods may exhibit toxicity in vivo and exhibit other side effects have. The bioadhesive of the present invention can be applied to tissues exposed after surgery to prevent adhesion occurring between the tissues and the surrounding tissues.

In another aspect, the present invention relates to a tissue engineering support comprising a hydrogel based on mussel adhesive protein.

The tissue engineering technique refers to culturing the cells isolated from a patient's tissue on a support to prepare a cell-support complex, and then transplanting the prepared cell-support complex into the body. The tissue engineering technique includes artificial skin, artificial bone, Cartilage, artificial cornea, artificial blood vessels, and artificial muscles. The bioadhesive hydrogel of the present invention can provide scaffolds similar to biotissues to optimize the regeneration of living tissues 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.

The hydrogel of the present invention facilitates the growth and differentiation of cells through interaction with cells or tissues of the human body, and various physiologically active substances which are involved in the function of regenerating and regenerating tissues can be easily attached. The physiologically active substance is also collectively referred to as various biomolecules that may be included to realize an artificial extracellular matrix having a structure similar to a natural extracellular matrix. Examples of the physiologically active substance include cells, proteins, nucleic acids, sugars, enzymes and the like, and examples thereof include cells, proteins, polypeptides, polysaccharides, monosaccharides, oligosaccharides, fatty acids and nucleic acids. have. 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 plasmid nucleic acid as a nucleic acid material, hyaluronic acid, heparin sulfate, chondroitin sulfate, an alginate salt as a saccharide substance, and a hormone protein as a protein substance.

In another aspect, the present invention relates to a carrier for drug delivery comprising a hydrogel based on mussel adhesive protein.

The bio-injectable tissue adhesive hydrogel according to the present invention can be used as an extracellular matrix as a scaffold for drug delivery. The drug is not particularly limited and includes a chemical substance, a small molecule, a peptide or a protein drug, a nucleic acid, a virus, an antibacterial agent, an anticancer agent, or an anti-inflammatory agent.

The small molecule may be, but is not limited to, a contrast agent (e.g., a T1 contrast agent, a T2 contrast agent such as a supernatant, a radioactive isotope, etc.), a fluorescent marker,

The peptide or protein drug may be a hormone, a hormone analogue, an enzyme, an enzyme inhibitor, a signaling protein or a portion thereof, an antibody or a portion thereof, a single chain antibody, a binding protein or a binding domain thereof, Proteins, cytokines, transcription factors, blood coagulation factors and vaccines, and the like. More specifically, a fibroblast growth factor (FGF), a vascular endothelial growth factor (VEGF), a transforming growth factor (TGF), a bone morphogenetic protein (HGF), pGH, leucocyte growth factor (G-CSF), erythrocyte growth factor (EPO), macrophage growth factor (M-CSF), tumor necrosis factor (TNF) (EGF), platelet-derived growth factor (PDGF), interferons, interleukins, calcitonin, NGF, growth hormone releasing factor, angiotensin, luteinizing hormone releasing hormone (LHRH) (LHRH agonist), insulin, thyroid stimulating hormone releasing hormone (TRH), angiostatin, endostatin, somatostatin, glucagon, endorphin, bacitracin, mergein, cholestin, monoclonal antibodies, vaccines or the like Lt; RTI ID = 0.0 > , But it is not limited thereto.

The nucleic acid may be, for example, RNA, DNA or cDNA, and the sequence of the nucleic acid may be an encoding site sequence or an unencrypted site sequence (e.g., an antisense oligonucleotide or an siRNA).

The virus may be a whole virus or a virus core comprising the nucleic acid of the virus (i.e., the nucleic acid of the packaged virus without the envelope of the virus). Examples of viruses and viral cores that may be carried include, but are not limited to, papilloma virus, adenovirus, baculovirus, retroviral core, and semilk virus core.

Wherein the antimicrobial agent is selected from the group consisting of minocycline, tetracycline, oproxacin, phosphomycin, mergein, proproxacin, ampicillin, penicillin, doxycycline, thienamycin, cephalosporin, noradocin, gentamicin, neomycin, , Pyromomycin, micronomycin, amikacin, tobramycin, dibecasin, cytotoxin, sepracur, erythromycin, cyprofloxacin, levofloxacin, enoxasin, vancomycin, imipenem, fosidic acid and mixtures thereof But is not limited thereto.

Wherein the anticancer agent is selected from the group consisting of paclitaxel, texothier, adriamycin, endostatin, angiostatin, mitomycin, bleomycin, cisplatin, carboplatin, doxorubicin, daunorubicin, dirubicin, 5-fluorouracil, methotrexate, Thymomycin-D, and mixtures thereof.

Wherein the anti-inflammatory agent is selected from the group consisting of acetaminophen, aspirin, ibuprofen, diclofenac, indomethacin, piroxycam, fenoprofen, plubiprofen, ketoprofen, naproxen, , ≪ / RTI > tenoxycam, and mixtures thereof.

Since the hydrogel based on the mussel adhesive protein provided by the present invention has excellent bioadhesion, it can be widely applied as a biocide adhesive, a tissue engineering support, or a drug delivery carrier.

Figure 1 shows the results of analysis of DOPA conversion in mussel adhesive protein fp-1 through an amino acid analyzer. The peak intensity of DOPA and tyrosine residues indicates a conversion rate of about 37%.
2 is an image of a hydrogel formed by oxidation of hydrogel and mussel adhesion protein fp-1 formed by binding of mussel adhesive protein fp-1 and Fe ^{3 +} , respectively.
FIG. 3 is a graph comparing the storage elasticity of a hydrogel formed by oxidation of hydrogel and mussel adhesive protein fp-1 formed by the combination of mussel adhesive protein fp-1 and Fe ^{3 +} .
FIG. 4 is a graph showing the results of comparative analysis of storage elasticity, loss elastic modulus, and loss tangent of a hydrogel formed by binding of mussel adhesive protein fp-1 and Fe ^{3 +} to a frequency of a hydrogel.
FIG. 5 shows the results of comparative analysis of storage elasticity, loss elastic modulus, and loss tangent of a hydrogel formed by oxidation of mussel adhesive protein fp-1 with a rheological device.
FIG. 6 shows the loss tangent value and the loss due to the self-recovery of the hydrogel formed by the oxidation of the hydrogel and the mussel adhesive protein fp-1 formed by the combination of the mussel adhesive protein fp-1 and Fe ^{3 +} Tangent values were measured by a rheometer, and the results were compared and analyzed.
FIG. 7 is an image showing that the binding number of DOPA residues and Fe ^{3 +} contained in the mussel adhesive protein fp-1 changes depending on pH through color change.
FIG. 8 shows the results of UV-vis spectroscopy analysis of color change of hydrogel formed by binding of mussel adhesive protein fp-1 and Fe ^{3 +} to pH.
FIG. 9 shows the results of analysis of color change of a hydrogel formed by oxidation of mussel adhesive protein fp-1 using UV-vis spectroscopy.
FIG. 10 shows the results of chemically confirming the binding of DOPA and Fe ^{3 +} according to pH in a hydrogel formed by binding of mussel adhesive protein fp-1 and Fe ^{3 +} using a Raman spectroscope.
Fig. 11 shows the results of measurement of the tissue adhesion force of a hydrogel formed by binding of mussel adhesive protein fp-1 and Fe ^{3 +} through pig skin using a tensile strength machine.
FIG. 12 shows the results of measuring the tissue adhesive force of a hydrogel formed by oxidation of mussel adhesive protein fp-1 in pig skin through a tensile strength machine.
FIG. 13 shows the result of continuously measuring the adhesive force of the hydrogel formed by the binding of the mussel adhesive protein fp-1 and Fe ^{3 +} to the self-resisting force.

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

Example  1. Based on mussel adhesive protein Hydrogel  Produce

1-1. Recombinant mussel adhesive protein fp -One variant Production and DOPA  Crystal reaction

The mussel adhesive protein fp-1 variant (SEQ ID NO: 3) used in the present invention was obtained by repeating the peptide consisting of AKPSYPPTYK in the amino acid sequence of mushroom adhesive protein fp-1 (Genbank No. Q27409; SEQ ID NO: 1) The fp-1 variant was produced in E. coli. 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.

The tyrosinase enzyme (mushroom tyrosinase, SIGMA) was used to modify the tyrosine amino acid, which constitutes the mussel adhesive protein fp-1 variant, 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. In order to analyze the modification efficiency of the mussel adhesive protein fp-1 variant, amino acid composition analysis showed that about 37% of the total tyrosine residues were converted to DOPA, and the results are shown in FIG.

1-2. Mussel adhesive protein fp -One variant Using Hydrogel  Manufacturing and Rheological  analysis

Two different chemical bonds were used to produce the hydrogel using the mussel adhesive protein fp-1 variant produced in Example 1-1. The first is the DOPA-metal coordination method using Fe ^{3 +} ions and the second is the DOPA-DOPA interaction method using the oxidation of NaIO ₄ .

Specifically, to prepare a hydrogel using the first method, a modified fp-1 variant was dissolved in PBS at a concentration of 30 to 50 wt%, and then a FeCl ₃ solution was added so that the ratio of DOPA: Fe ^{3 +} was 3: 1 . The gel was then formed by increasing the pH by adding 1N NaOH solution until the color became a deep purple color.

In order to prepare the hydrogel using the second method, the same modified fp-1 variant was dissolved in PBS at a concentration of 30 to 50 wt%, and NaIO ₄ The solution was added so that the ratio of DOPA: IO ₄ ^- was 2: 1, and the gel turned into dark reddish brown within 5 minutes.

A photograph of the formed gel is shown in Fig.

The rheometer was used to analyze the rheological properties of the gel.

Specifically, in order to compare the storage modulus with time of the hydrogel prepared by the above two methods, it is necessary to determine the storage modulus at 10% strain (0 to 900 sec) strain) at a frequency of 10 Hz, and the storage elastic modulus was measured. The results are shown in FIG. As shown in FIG. 3, it can be seen that the method using the Fe ^{3 +} ion has already formed a certain amount of gel at the same time as the pH is elevated, and that the method of oxidizing gradually forms a gel gradually with time.

Next, in order to analyze the storage elastic modulus and loss modulus according to the change of frequency with keeping the strain constant, the 20% strain is maintained and the storage elastic modulus and the loss elastic modulus are changed while varying the frequency from 0.1 to 100 Hz The loss tangent delta value was determined. The gel preparation method using Fe ^{3 +} ion was shown in FIG. 4, and the gel preparation method using oxidation was shown in FIG. 5. As can be seen from FIG. 4, the gel using Fe ³⁺ ion exhibited a fluid gel shape that can easily change its shape at low frequencies. As shown in FIG. 5, stiff gel shape.

In order to investigate whether each gel can be self-healed with the original strength after breaking the gel with high strain, the strain was changed from 1 to 1000% while keeping the frequency at 80 Hz, After the values were analyzed, the loss tangent values over time were analyzed while lowering to a strain of 10%. As a result, as shown in FIG. 6, it was found that the original strength was restored in the case of the gel using Fe ^{3 +} ions, and it was confirmed that the gel caused by the oxidation showed lower strength than the original strength. In addition, the self-resilience of the gel using Fe ^{3 +} ions was also confirmed by a continuous tissue adhesion test, and the results are shown in FIG.

Example  2. Mussel adhesive protein fp -One variant Using Hydrogel  Analysis of chemical bonding process involved in formation

The chemical binding process involved in the hydrogel formation using the variant of mussel adhesive protein fp-1 prepared in Example 1-2 was observed by UV-Visible spectroscopy and analyzed by Raman spectroscopy.

Specifically, the modified fp-1 variant was dissolved in PBS at a concentration of 4 mg / ml, and the FeCl ₃ solution was added thereto in the same manner as in Example 1-2. And the result of analysis by UV-Visible spectroscopy is shown in FIG. As a result, it showed a green color indicating mono bonding of DOPA and Fe ^{3 +} at pH 4.0 or lower, a blue series showing bis bonding at pH 5.5 and a deep pink series showing tris bonding at pH 8.0 and above Respectively.

Further, as shown in FIG. 9, in the same manner as in <Example 1-2>, NaIO ₄ When the solution was added, it was confirmed to be reddish brown indicating DOPA quinine type.

In order to more chemically confirm the binding pattern between DOPA and Fe ^{3 +} in the hydrogel using Fe ^{3 +} ion, the spectrum according to pH was analyzed using Raman spectroscopy. As the pH increased, DOPA and Fe ^{3 +} And the intensity of the peak due to the coupling of the peak is increased, which is shown in Fig.

Example  3. Mussel adhesive protein fp -One variant Using Hydrogel  Biocompatibility

A hydrogel was prepared using the variant of mussel adhesive protein fp-1 according to the method of Example 1-2, and adhesion test was performed to see how much the hydrogel had adhesive strength on a surface similar to the actual tissue. At this time, the gelation conditions were such that the ratio of DOPA: Fe ³⁺ was 3: 1 and DOPA: IO ₄ ^- was 2: 1.

Specifically, pig skin-derived skin samples (Stellen Medical) from which fats were removed were immersed in PBS and immobilized with a cyanoacrylate bond on aluminum specimens, and then treated with hydrogel between skin samples. After the hydrogel was treated and the skin samples were bonded, the hydrogel using Fe ^{3 +} was added to each buffer pH, and the hydrogel using NaIO ₄ was allowed to stand in a dry state. The crosslinking reaction was allowed to proceed for 2 hours. Thereafter, the adhesive strength was measured using a tensile strength meter (Instron).

As a result, it was confirmed that the hydrogel using Fe ^{3 +} has an increased adhesive force as the crosslinking buffer pH increases, which is shown in FIG. It was confirmed that the hydrogel using NaIO ₄ improves the adhesion as the crosslinking time in the dry state is increased, which is shown in FIG.

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Mussel adhesive protein-based adhesive hydrogel <130> DPP20133947 <160> 25 <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> 120 <212> PRT <213> Artificial Sequence <220> <223> fp-1 variant <400> 3 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> 4 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> fp-1 variant-RGD <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 Gly Arg Gly Asp Ser Pro         115 120 125 <210> 5 <211> 456 <212> PRT <213> Artificial Sequence <220> <223> fp-2 <400> 5 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> 6 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> fp-3 <400> 6 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> 7 <211> 787 <212> PRT <213> Artificial Sequence <220> <223> fp-4 <400> 7 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> 8 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> fp-5 <400> 8 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> 9 <211> 99 <212> PRT <213> Artificial Sequence <220> <223> fp-6 <400> 9 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> 10 <211> 196 <212> PRT <213> Artificial Sequence <220> <223> fp-151 <400> 10 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> 11 <211> 202 <212> PRT <213> Artificial Sequence <220> <223> fp-151-RGD <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 Gly Arg Gly Asp Ser Pro         195 200 <210> 12 <211> 172 <212> PRT <213> Artificial Sequence <220> <223> fp-131 <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 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> 13 <211> 175 <212> PRT <213> Artificial Sequence <220> <223> fp-353 <400> 13 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> 14 <211> 189 <212> PRT <213> Artificial Sequence <220> <223> fp-153 <400> 14 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> 15 <211> 189 <212> PRT <213> Artificial Sequence <220> <223> fp-351 <400> 15 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> 16 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 1 <400> 16 Arg Gly Asp   One <210> 17 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 2 <400> 17 Arg Gly Asp Ser   One <210> 18 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 3 <400> 18 Arg Gly Asp Cys   One <210> 19 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 4 <400> 19 Arg Gly Asp Val   One <210> 20 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 5 <400> 20 Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro   1 5 10 <210> 21 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 6 <400> 21 Gly Arg Gly Asp Ser   1 5 <210> 22 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 7 <400> 22 Gly Arg Gly Asp Thr Pro   1 5 <210> 23 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 8 <400> 23 Gly Arg Gly Asp Ser Pro   1 5 <210> 24 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 9 <400> 24 Gly Arg Gly Asp Ser Pro Cys   1 5 <210> 25 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 10 <400> 25 Tyr Arg Gly Asp Ser   1 5

Claims (16)

Wherein the amino acid sequence of SEQ ID NO: 2 comprises 1 to 12 consecutive recombinant mussel adhesive proteins,
A bioadhesive hydrogel having an adhesive force in a living tissue of 50 to 200 kPa.
The hydrogel according to claim 1, wherein the hydrogel is formed from a combination of a 3,4-dihydroxyphenylalanine residue and Fe ³⁺ ions contained in the recombinant mussel adhesive protein.
The hydrogel according to claim 1, wherein the hydrogel is formed by oxidizing a waveguide contained in a recombinant mussel adhesive protein.
2. The hydrogel according to claim 1, wherein the recombinant mussel adhesive protein is at least 20% of the total tyrosine residues are waveguide-modified.
delete delete delete The recombinant mussel adhesive protein according to claim 1, wherein the recombinant mussel adhesive protein is a protein consisting of the amino acid sequence of SEQ ID NO: 3.
2. The hydrogel according to claim 1, wherein the recombinant mussel adhesive protein is a polypeptide having a carboxyl terminal or a terminal amino terminal with a polypeptide consisting of 3 to 25 amino acids including RGD (Arg Gly Asp).
10. The hydrogel according to claim 9, wherein the polypeptide comprising RGD is an amino acid sequence selected from the group consisting of SEQ ID NO: 16 to SEQ ID NO:
11. The hydrogel according to claim 10, wherein the recombinant mussel adhesive protein is a protein consisting of the amino acid sequence of SEQ ID NO: 4.
Wherein the amino acid sequence of SEQ ID NO: 2 is reacted with the recombinant mussel adhesion protein and the Fe &lt; ^{3 +} &gt;
A method for producing a bioadhesive hydrogel according to any one of claims 1 to 4 and 8 to 11.
Wherein the amino acid sequence of SEQ ID NO: 2 comprises 1 to 12 consecutive amino acids of the recombinant mussel adhesive protein.
A method for producing a bioadhesive hydrogel according to any one of claims 1 to 4 and 8 to 11.
A bioadhesive composition comprising the hydrogel according to any one of claims 1 to 4 and 8 to 11.
A tissue engineering support comprising the hydrogel of any one of claims 1 to 4 and 8 to 11.
A drug delivery carrier comprising the hydrogel according to any one of claims 1 to 4 and 8 to 11.

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