WO2018186702A1 - Multi-block copolypeptide, which is composed of elastin-based peptide and mussel foot protein and has stimulus responsivity and surface adhesiveness, preparation method therefor, and use thereof - Google Patents

Abstract

The present invention relates to a multi-block copolypeptide having stimulus responsivity and surface adhesiveness. When the multi-block copolypeptide composed of an elastin-based peptide and a mussel foot protein, of the present invention, is used, a self-assembled core-shell structure and a hydrogel, both of which can be reversibly changed according to temperature stimuli, can be formed, and the multi-block copolypeptide has remarkably excellent surface adhesiveness, thereby being usable in the field of biomedical applications.

Description

Multi-block copolypeptides comprising elastin-based peptides and mussel quartet proteins with stimulatory responsiveness and surface adhesion, and methods for their preparation and use thereof

The present invention relates to a multi-block copolypeptide having a stimulus responsiveness and surface adhesion, and more particularly, to a multi-block copolypeptide consisting of an elastin-based peptide and a mussel foot protein, a method of preparing the multi-block copolypeptide, A core-shell self-assembled nanostructure comprising a block copolypeptide and a hydrogel comprising the multi-block copolypeptide.

Self-assembly of protein-based copolymer blocks into micelle or hydrogel structures with responsiveness to environmental changes such as temperature, pH and ionic strength has been studied for decades because of their high biocompatibility and controllable resolution. Protein-based polypeptide blocks self-assembled into core-shell micelles have received considerable attention as drug delivery systems, and in particular, triple block polypeptides have been studied for tissue engineering applications because sol-gel transitions due to physical or chemical crosslinking occur. come. In addition, various protein-based materials have been developed for drug delivery and tissue engineering applications.

Bioadhesives refer to substances that have adhesion properties to various biological samples such as proteins, DNA, growth factors, cells, etc., such as cell membranes, cell walls, lipids, proteins, DNA, growth factors, cells, and tissues. Various biomedical applications are possible, such as engineering supports, drug delivery carriers, tissue fillers, wound healing, or intestinal adhesion prevention. Bioadhesives require strong adhesion and crosslinking capabilities and must maintain their function in vivo for a long time. Bioadhesives currently commercially available or commercially available include cyanoacrylate instant adhesives, fibrin glues, gelatin glues and polyurethane-based adhesives. However, the bioadhesives using synthetic polymers show very weak strength in the presence of an aqueous solution in vivo, and cyanoacrylate-based bioadhesives have been pointed out as causing great side effects such as immune responses in the human body. In addition, the fibrin-based bioadhesives currently used in actual patients have little side effects, but their use is limited because of their very low adhesion. In the case of gelatin tissue adhesive, formalin or glutaaldehyde, which is used as a crosslinking agent, also causes cross-linking reaction with proteins in vivo, resulting in tissue toxicity, and polyurethane-based tissue adhesive has a problem in that aromatic diisocyanate, which is a synthetic raw material, is biotoxic. .

Marine mussels, on the other hand, can inhabit a variety of aquatic surfaces in harsh environments due to their footwort proteins. Dopa (Dihydroxyphenylalanine (DOPA)) of mussel foot protein (MFP) plays an important role in surface adhesion. Mussels are classified into six types, from type 1 to type 6. Each MFP has DOPA residues in different content ratios, thus exhibiting varying surface adhesion. For example, MFP 1, 2, and 4 cross-molecularly and intramolecularly crosslink the proteins, while MFP 3, 5, and 6 exhibit surface adhesion by interaction with inorganic and organic molecules. Thus, DOPA makes important chemical contributions to adhesion through intermolecular and intramolecular crosslinking (Silverman HG et al., Marine biotechnology, 9 (6), 661-681, 2007; Lee Haeshin. Et al., Proceedings of the National Academy of Sciences, 103 (35), 12999-13003, 2006). Waveguides with catechol side chains in which tyrosine residues are hydroxylated by tyrosinase can bind metal ions, oxides and semimetals through coordination bonds or hydrogen bonds (Sever, MJ et al., Angewandte). Chemie, 116 (4), 454-456, 2004).

Recently, studies have been reported that block polypeptides composed of elastin-based peptides and resilin-like polypeptides have phase transfer behavior and can be self-assembled, but do not disclose surface adhesion. 2017-0113209).

Accordingly, the present inventors have made efforts to find a polypeptide having a stimulus responsiveness and surface adhesion that can be used in biomedical applications, elastin-based peptide (Elastin-based Polypeptide, EBP); And using a multiblock copolypeptide (MFP) consisting of mussel quartet protein (MFP), forms a self-assembled core-shell structure and hydrogel, reversible change with temperature stimulation, surface adhesion It confirmed that it was remarkably excellent, and completed this invention.

It is an object of the present invention to provide multiblock copolypeptides having stimulatory reactivity and surface adhesion.

Another object of the present invention is a gene encoding the multiblock copolypeptide, a recombinant vector comprising the gene, a recombinant microorganism into which the gene or the recombinant vector is introduced, and a method for producing a multiblock copolypeptide using the recombinant microorganism. To provide.

Still another object of the present invention is a self-assembling nanostructure and a self-assembling nanostructure of the core-shell structure in which the multiblock copolypeptide is a temperature structure, the EBP block forms a core structure, and the MFP block forms a shell structure. To provide a drug delivery composition comprising a structure.

Still another object of the present invention is to prepare a multi-block copolypeptide cross-linking between block polypeptides by temperature stimulation, hydrogel, bioadhesive composition comprising the hydrogel and surgery comprising the hydrogel To provide a suture.

In order to achieve the above object, the present invention provides an elastin-based peptide (EBP); And multi-block copolypeptides consisting of mussel foot protein (MFP).

The present invention also provides a gene encoding the multiblock copolypeptide, a recombinant vector comprising the gene, a recombinant microorganism into which the gene or the recombinant vector is introduced.

The present invention also comprises the steps of (a) culturing a recombinant microorganism to produce the multi-block copolypeptide; And (b) obtaining the generated multiblock copolypeptide.

The present invention also provides a self-assembled nanostructure and a self-assembled nanostructure of the core-shell structure wherein the multi-block copolypeptide is a temperature structure, the EBP block forms a core structure, the MFP block forms a shell structure It provides a drug delivery composition comprising.

The present invention also provides a hydrogel in which the multiblock copolypeptides are prepared by forming crosslinks between block polypeptides by temperature stimulation.

The present invention also provides a bioadhesive composition and a surgical suture containing the hydrogel.

The multiblock copolypeptides of the present invention form self-assembled core-shell structures and hydrogels that can be reversibly changed with temperature stimulation, and have excellent surface adhesion, which makes them useful in biomedical applications. Can be.

1 is a molecular schematic of various block copolypeptides composed of EBP and MFP ((A): double and triple block copolypeptides forming a core-shell (micelle) structure, (B): MFP forming a hydrogel) -EBP-MFP triple block copolypeptide, (C): EBP-MFP-EBP triple block copolypeptide to form hydrogel, (D): Stimulation responsiveness and surface adhesion of EBP-MFP-EBP triple block copolypeptide Sex mechanism).

Figure 2 is a result of confirming the MFP DNA of the present invention by agarose gel electrophoresis ((A): Mcfp5, (B) Mgfp5, lane (M): size marker, lane (1): 1 MFP repeat unit, lane (2): 2 MFP repeat units, lane (3): 4 MFP repeat units).

Figure 3 illustrates the cloning of the EBP-MFP block copolypeptide gene of the present invention ((A): multiplex cloning of MFP, (B): EBP-MFP block copolypeptide cloning).

4 is a schematic of the cloning of the tyrosinase and ORF438 genes of the present invention ((A): tyrosinase cloning, (B): ORF438 cloning).

Figure 5 shows the results of (A) tyrosinase and (B) ORF438DNA of the present invention by agarose gel electrophoresis (lane (1) and (2) DNA template: 25ng, lane (3) and (4) DNA Template: 50 ng, lane (N): negative control).

FIG. 6 shows (A) DNA agarose gel electrophoresis (1.2%) of tyrosinase gene (824bp) and orf438 gene (438bp); (B) SDS-PAGE results of tyrosinase (˜35 kDa), EBP-MFP double block copolypeptide (˜24 kDa) and orf438 (˜15 kDa) co-expressed in E. coli (lane (1): EBP-MFP double block) Copolypeptide, lane (2): tyrosinase and orf438, lane (3): double block copolypeptide, tyrosinase and orf438 co-expressed in E. coli); (C) SDS-PAGE results of EBP-MFP-EBP triple block copolypeptide (˜41 kDa), tyrosinase (˜35 kDa) and orf438 (˜15 kDa) co-expressed in E. coli (lane (1): EBP-MFP) -EBP triple block copolypeptide, lane (2): triple block copolypeptide, tyrosinase and orf438 co-expressed (coexpressed) in E. coli; (D) Schematic representation of the chemical mechanism of EBP-MFP block copolypeptide tyrosine modification; (E) NBT staining hydroxylation via a simultaneous expression system of tyrosinase, ORF438 and double block copolypeptides in E. coli of EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptides The result is confirmed. (F) EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ -EBPPI [G ₁ A ₄ F ₁ ] ₆ Triple Block Copolypeptides of Tyrosinase, ORF438 and Triple Block Copolypeptides in Escherichia coli Hydroxylation through the co-expression system was confirmed by NBT staining.

Figure 7 is a result of NBT / Glycinate staining after treatment with mushroom-derived tyrosinase in order to confirm the tyrosine residue modification of the double block copolypeptide of the present invention. (1) Dopamine hydrochloride as a positive control, (2) EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ treated with mushroom-derived tyrosinase, (3) as a negative control Unencoded EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ , (4) EBPPI [G ₁ A ₄ F ₁ ] ₆ )

8 shows SDS-PAGE results of copper staining of the block copolypeptide of the present invention ((A): EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block treated with mushroom-derived tyrosinase. Copolypeptide, (B): EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ -EBPPI [G ₁ A ₄ F ₁ ] ₆ triple block copolypeptide treated with mushroom derived tyrosinase, ( C): EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptides treated with various concentrations of NaIO ₄ (Lane (M): standard protein marker, Lane (1): EBPPI [G) ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ , Lane (2): EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ , Lane (3): 50 mM treated with 5 mM NaIO ₄ a EBPPI treated with _{NaIO 4 [4 G 1 a F} 1] 6 -MFP [Mgfp5] 1, Lane (4): hydroxylation of _{EBPPI [G 1 a 4 F 1} ] 6 -MFP [Mgfp5] 1, Lane ( 5): 5 mM NaIO hydroxylation EBPPI the _four treated with _{[G 1 a 4 F 1]} 6 -MFP [Mgfp5] 1, Lane (6): 50 mM NaIO 4 by hydroxylation of EBPPI [G treated with ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] _1. Dual and To modify the tyrosine residues of the triple block copolypeptides, mushroom derived tyrosinase was treated.

9 is a result of confirming the thermal profile according to each condition ((A) (a): mushroom-derived tyrosinase-treated EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptide , (b): unmodified EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptide and (c): control EBPPI [G ₁ A ₄ F ₁ ] ₆ monoblock; ( B) Mushroom-derived tyrosinase treated EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ according to the double block copolypeptide concentration; (C) Mushroom-derived tyrosinase according to the concentration of NaIO ₄ Treated EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ ; (D) 25 μM concentration of mushroom-derived tyrosinase treated EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block And mushroom-derived tyrosinase-treated EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ -EBPPI [G ₁ A ₄ F ₁ ] ₆ triple block copolypeptides).

(A) Photograph of mushroom-derived tyrosinase-catalyzed EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptide treated with various concentrations (10-100 mM) of NaIO ₄ ; (B) a gel photograph of 30 wt% double block copolypeptides treated with 100 mM NaIO ₄ ; (C) Photograph of 40% by weight double block copolypeptide treated with 10 mM NaIO ₄ .

FIG. 11 shows (A) mushroom-derived tyrosinase-catalyzed EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ -EBPPI [G ₁ A ₄ F ₁ ] ₆ triple block nose treated with ₁₀ mM NaIO ₄ Polypeptide (10% by weight) photo; (B) The photo on the left shows mushroom-derived tyrosinase-catalyzed EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ -EBPPI [G ₁ A ₄ F ₁ ] ₆ triple block under (A) conditions Surface attachment picture of the copolypeptide, the right is a 20% by weight EBP triple block copolypeptide picture as a control treated with (A) conditions.

FIG. 12 is a photograph of 10 mM NaIO ₄ treated with hydroxylated block copolypeptide (10 wt.%) In a coexpression system; (B) Photos of adhesion experiments in the presence of water of hydroxylated blockcopolypeptides in a coexpression system.

FIG. 13 was measured by the DLS instrument with the hydrodynamic radius of (A) mushroom-derived tyrosinase-catalyzed hydroxylation with or without modification. The hydrodynamic radius of the block copolypeptides was measured at 12.5 uM in 10 mM phosphate buffer (pH 5). The hydrodynamic radius of the block copolypeptides after phase transition is 50 nm to 70 nm, indicating that the block copolypeptides are in the form of specific structures. ((a): unmodified EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptide at 10 ^o C, (b): unmodified EBPPI [G at 45 ^o C ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptide, (c): Mushroom-derived tyrosinase treated EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] at 10 ^o C ₁ double block copolypeptide and (d): mushroom-derived tyrosinase treated EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptide at 45C) (B) in a coexpression system Surface adhesion of the hydroxylated EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptide was confirmed by fluorescent dyes. (1) Confirmation of non-specific reaction between surface of tube and fluorescent dye, (2) Confirmation of surface adhesion between EBP double block copolypeptide and fluorescent dye, (3) Hydroxylated EBPPI [G ₁ A ₄ F ₁ ] ₆ Confirming surface adhesion below the transition temperature of -MFP [Mgfp5] ₁ double block copolypeptide, (4) hydroxylated EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptide Of surface adhesion above the transition temperature of

FIG. 14 shows transmission electron micrographs of the temperature of EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptides with mushroom-derived tyrosinase-catalyzed hydroxylation ((A, B , C) nanostructures of EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptides observed at 10 ^° C., with accumulations of 0.2 μm, 50 nm and 50 nm, respectively (D, E , F) constructs of EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptides observed at 37 ^° C., with accumulations of 0.5 mm, 100 nm and 50 nm, respectively (G, H, I) The structure of EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptides observed at 50 ^° C., with accumulation of 0.5um, 100nm and 50nm, respectively.

In the present invention, elastin-based peptide (EBP); And multiblock copolypeptides (MFPs) of mussels form a self-assembled core-shell structure and hydrogel that can be reversibly changed with temperature stimulation and have excellent surface adhesion. It was confirmed that.

Thus, in one aspect, the present invention provides an elastin-based peptide (EBP); And it relates to a multiblock copolypeptide (multiblock copolypeptide) consisting of mussels musa protein (MFP).

In the present invention, the term "copolypeptide" means a polypeptide that is a copolymer.

In the present invention, the term "polypeptide" means any polymer chain of amino acids. The terms "peptide" and "protein" are interchangeable with the term "polypeptide", which also refers to a polymer chain of amino acids. The term "polypeptide" includes "polypeptide" analogs of natural or synthetic proteins, protein fragments and protein sequences. The polypeptide may be a monomer or a polymer.

The term "phase transition" means that the state of a substance changes, such as when water turns into water vapor or ice turns into water.

Polypeptides having the phase change behavior according to the present invention are basically stimulin reactive elastin-based polypeptides (EBPs). The "elastin-based polypeptides" are also called "elastin-like polypeptides (ELPs)." It is a term widely used in the technical field of the present invention.

The EBP undergoes a reversible phase transition at lower critical solution temperature (LCST), also referred to as transition temperature (T _t ). These have large water solubility below T _t , but become insoluble when the temperature exceeds T _t .

In the present invention, the physicochemical properties of EBP are mainly controlled by the combination of pentapeptide repeat units Val-Pro- (Gly or Ala) -X _aa -Gly [VP (G or A) XG]. Specifically, the third amino acid of that repeat unit determines the relative mechanical properties. For example, in the present invention, the third amino acid Gly determines elasticity, or Ala determines plasticity. The elasticity or plasticity is a property that appears after the transition.

On the other hand, both the hydrophobicity and the multimerization of the pentapeptide repeating unit of the guest residue X _{aa as} the fourth amino acid affect T _t .

Mussel triglyceride protein of the present invention can be attached to various surfaces through waveguide. DOPA having a catechol side chain imparts surface adhesion through hydrogen bonds and coordinate bonds with surface molecules, and quinones, an oxidized form of DOPA, exhibit cohesion through intermolecular crosslinking. Since quinones are an oxidized form of DOPA, they cannot interact with surface molecules, but they can be reacted with aryl-aryl coupling, metal chelation, or Michael-type addition reactions with amine-containing proteins. Intramolecular and intermolecular crosslinking forms to provide strong underwater cohesion. Crosslinking through quinone formation produces a hardened sheath and exhibits moisture-resistance. Therefore, DOPA and quinone are essential for surface adhesion, which are determined by pH conditions. When mussels secrete MFP for surface attachment, the pH conditions around mussel quiescents are lower than pH 3.0, limiting the oxidation of DOPA to adsorb to surface oxides through hydrogen bonding and metal ion coordination. After surface attachment, MFP is exposed to seawater (pH-8.3) to induce oxidation from DOPA to quinones, crosslinking and protein coagulation. In addition, hydrophilic amino acids of MFPs such as Ser and Gly participate in cohesive interactions by hydrogen bonding, cation-pi interactions, electrostatic and hydrophobic interactions (Waite, JH, Journal of Experimental) Biology, 220 (4), 517-530, 2017).

In the present invention, a novel type of block copolypeptide consisting of a multifunctional EBP block and an MFP block is reasonably designed, synthesized, and characterized. Based on the surface adhesion of the MFP domain in nature, the present invention was intended to combine with MFP and EBP block, a stimulatory reactive protein, to observe self-assembly structure with biomimetic underwater adhesion, and to apply it to the biomedical field. To achieve strong interfacial underwater adhesive properties, the mussel californianus foot protein 5 (Mcfp5) and the Mediterranean have a high percentage of tyrosine content (~ 30%) in all mussel foot protein types. The gene sequence of mussel galloprovincialis foot protein 5 (Mgfp5) was selected. Tyrosine content of MFP is related to surface adhesion efficiency and strength (Silverman H. G. et al., Marine biotechnology, 9 (6), 661-681, 2007).

In the present invention, the multiblock copolypeptide is in the group consisting of (EBP) n (MFP) n, (EBP) n (MFP) n (EBP) n and (MFP) n (EBP) n (MFP) n It is composed of any one arrangement, wherein n is an integer of 1 or more, it may be characterized in that the repetition number of EBP or MFP.

In the present invention, the elastin-based peptide (EBP) is selected from any one of a group consisting of [VPGXG VPGXG VPGXG VPGXG VPGXG VPGXG] block, [VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG] block and [IPAXG IPAXG IPAXG IPAXG IPAXG IPAXG] block. It is composed of the amino acid sequence represented by the formula, X may be characterized in that the amino acid except proline.

In the present invention, X (or X _aa ) is referred to as a "guest residue". Various kinds of X _aa may be introduced to prepare various kinds of EBP according to the present invention.

The polypeptide may have multi-stimuli responsiveness.

The term "multi-stimulatory responsiveness" means to be responsive to one or more stimuli. Specifically, the stimulus may be one or more selected from the group consisting of temperature, pH, ionic strength and ligand.

Ligand in the present invention is a substance that specifically binds to a desired substance, for example, various antibodies, antigens, enzymes, substrates, receptors, peptides, DNA, RNA, aptamers, protein A, protein G, avidin, Biotin, chelate compounds, various metal ions (e.g., calcium ions) and the like.

In the present invention, the [VPGXG VPGXG VPGXG VPGXG VPGXG VPGXG] block is characterized by the amino acid sequence of SEQ ID NO: 1, wherein the [VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG] block is represented by the amino acid sequence of SEQ ID NO: 2 And the [IPAXG IPAXG IPAXG IPAXG IPAXG IPAXG] block is represented by the amino acid sequence of SEQ ID NO.

In the present invention, the term "amino acid" means a natural "amino acid" or "artificial" amino acid, and preferably means a natural "amino acid". For example, the amino acid refers to glycine, alanine, serine, valine, leucine, isoleucine, methionine, glutamine, asparagine, cysteine, histidine, phenylalanine, arginine, tyrosine or tryptophan.

The nature of these amino acids is well known in the art. Specifically, it shows hydrophilicity (negative charge or positive charge) or hydrophobicity, and also shows the properties of aliphatic or aromatic.

Abbreviations such as Gly (G) and Ala (A) used in the present invention are amino acid abbreviations. Amino acid abbreviations are glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), proline (Pro, P), phenylalanine (Phe, F ), Tyrosine (Tyr, Y), tryptophan (Trp, W), cysteine (Cys, C), methionine (Met, M), serine (Ser, S), threonine (Thr, T), lysine (Lys, K) It is represented by arginine (Arg, R), histidine (His, H), aspartic acid (Asp, D), glutamic acid (Glu, E), asparagine (Asn, N), glutamine (Gln, Q). The abbreviation is a widely used expression in the art.

In the present invention, "hydrophilic amino acid" is an amino acid exhibiting hydrophilic properties, such as lysine, arginine, and the like, "hydrophobic amino acid" is an amino acid showing hydrophobic properties, such as phenylalanine, leucine and the like.

In the present invention, X in the [VPGXG VPGXG VPGXG VPGXG VPGXG VPGXG] block is A (Ala), G (Gly), I (Ile) in a ratio of 1: 4: 1; K (Lys), G (Gly), I (Ile) are in a ratio of 1: 4: 1; Or D (Asp), G (Gly), I (Ile) in a ratio of 1: 4: 1; E (Glu), G (Gly), I (Ile) consists of a ratio of 1: 4: 1; G (Gly), A (Ala), F (Phe) consists of 1: 3: 2 ratio; K (Lys), A (Ala), F (Phe) consists of a ratio of 1: 3: 2; D (Asp), A (Ala), F (Phe) consists of a ratio of 1: 3: 2; K (Lys), F (Phe) consist of 3: 3 ratio; D (Asp), F (Phe) consist of 3: 3 ratio; H (His), A (Ala), I (Ile) are in a ratio of 3: 2: 1; H (His), G (Gly) is in the ratio of 5: 1; Or G (Gly), C (Cys), F (Phe) in a ratio of 1: 3: 2.

In the present invention, X in the [VPAXG VPAXG VPAXG VPAXG VPAXG VPAX] block is A (Ala), G (Gly), I (Ile) in a ratio of 1: 4: 1; K (Lys), G (Gly), I (Ile) are in a ratio of 1: 4: 1; Or D (Asp), G (Gly), I (Ile) in a ratio of 1: 4: 1; E (Glu), G (Gly), I (Ile) consists of a ratio of 1: 4: 1; Or G (Gly), A (Ala), F (Phe) in a ratio of 1: 3: 2.

In the present invention, X in the [IPAXG IPAXG IPAXG IPAXG IPAXG IPAXG] block is characterized by G (Gly), A (Ala), and F (Phe) in a ratio of 1: 4: 1 or 1: 3: 2. You can do

In the present invention, different EBPs having a pentapeptide repeating unit Val-Pro- (Gly or Ala) -X _aa -Gly [VP (G or A) XG] are named as follows. X _aa may be any amino acid except Pro. First, the pentapeptide repeat of the plastic Val-Pro-Ala-X _aa- Gly (VPAXG) is defined as the plastic elastin-based polypeptide with plasticity (EBPP). Meanwhile, the pentapeptide repeat of Val-Pro-Gly-X _aa -Gly (VPGXG) is called an elastin-based polypeptide with elasticity (EBPE). The pentapeptide repeat of Ile-Pro-Ala-X _aa- Gly (IPAXG) is also defined as a plastic elastin-based polypeptide (EBPPI) with the first position replaced by Ile. In [XiYjZk] n, the capital letters in parentheses are the short-term amino acid code of the guest residue, ie, the amino acid at position 4 (X _aa or X) of the EBP pentapeptide, and their corresponding subscripts are EBP monomers as repeat units. Shows the ratio of guest residues in the gene. The subscript number n of [XiYjZk] n is SEQ ID NO: 1 [VPGXG VPGXG VPGXG VPGXG VPGXG VPGXG], SEQ ID NO: 2 [VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG], or SEQ ID NO: 3 [IPAXG IPAXG IPAXG IPAXG IPAXG] The total number of repetitions of the EBP is shown. For example, EBPP [G1A3F2] 12 is an EBPP block consisting of 12 repeating units of SEQ ID NO: 2 [VPAXG VPAXG VPAXG VPAXG VPAXG], wherein Gly, Ala, and at the fourth guest residue position (X _aa ) The ratio of Phe is 1: 3: 2.

The gene and amino acid sequences of the EBP block of the present invention are shown in Tables 1 and 2, respectively.

Gene sequence of the EBP library

EBPE [A 1 G 4 I 1 ] (SEQ ID NO: 4)	GTC CCA GGT GGA GGT GTA CCC GGC GCG GGT GTC CCA GGT GGA GGTGTA CCT GGG GGT GGG GTC CCT GGT ATT GGC GTA CCT GGA GGC GGC
EBPP [A 1 G 4 I 1 ] (SEQ ID NO: 5)	GTT CCA GCT GGC GGT GTA CCT GCT GCT GCT GTT CCG GCC GGT GGTGTT CCG GCG GGC GGC GTG CCT GCA ATA GGA GTT CCC GCT GGT GGC
EBPE [K 1 G 4 I 1 ] (SEQ ID NO: 6)	GTT CCG GGT GGT GGT GTT CCG GGT AAA GGT GTT CCG GGT GGT GGTGTT CCG GGT GGT GGT GGT GTT CCG GGT ATC GGT GTT CCG GGT GGC
EBPP [K 1 G 4 I 1 ] (SEQ ID NO: 7)	GTT CCG GCG GGT GGT GTT CCG GCG AAA GGT GTT CCG GCG GGT GGTGTT CCG GCG GGT GGT GTT CCG GCG ATC GGT GTT CCG GCG GGT GGC
EBPE [D 1 G 4 I 1 ] (SEQ ID NO: 8)	GTT CCG GGT GGT GGT GTT CCG GGT GAT GGT GTT CCG GGT GGT GGTGTT CCG GGT GGT GGT GGT GTT CCG GGT ATC GGT GTT CCG GGT GGC
EBPP [D 1 G 4 I 1 ] (SEQ ID NO: 9)	GTT CCG GCG GGT GGT GTT CCG GCG GAT GGT GTT CCG GCG GGT GGTGTT CCG GCG GGT GGT GTT CCG GCG ATC GGT GTT CCG GCG GGT GGC
EBPE [E 1 G 4 I 1 ] (SEQ ID NO: 10)	GTT CCG GGT GGT GGT GTT CCG GGT GAA GGT GTT CCG GGT GGT GGTGTT CCG GGT GGT GGT GGT GTT CCG GGT ATC GGT GTT CCG GGT GGC
EBPP [E 1 G 4 I 1 ] (SEQ ID NO: 11)	GTT CCG GCG GGT GGT GTT CCG GCG GAA GGT GTT CCG GCG GGT GGTGTT CCG GCG GGT GGT GTT CCG GCG ATC GGT GTT CCG GCG GGT GGC
EBPE [G 1 A 3 F 2 ] (SEQ ID NO: 12)	GTC CCG GGT GCG GGC GTG CCG GGA TTT GGA GTT CCG GGT GCG GGTGTT CCA GGC GGT GGT GTT CCG GGC GCG GGC GTG CCG GGC TTT GGC
EBPP [G 1 A 3 F 2 ] (SEQ ID NO: 13)	GTG CCG GCG GCG GGC GTT CCA GCC TTT GGT GTG CCA GCG GCG GGAGTT CCG GCC GGT GGC GTG CCG GCA GCG GGC GTG CCG GCT TTT GGC
EBPP [K 1 A 3 F 2 ] (SEQ ID NO: 14)	GTG CCG GCG GCG GGC GTT CCA GCC TTT GGT GTG CCA GCG GCG GGAGTT CCG GCC AAA GGC GTG CCG GCA GCG GGC GTG CCG GCT TTT GGC
EBPP [D 1 A 3 F 2 ] (SEQ ID NO: 15)	GTG CCG GCG GCG GGC GTT CCA GCC TTT GGT GTG CCA GCG GCG GGAGTT CCG GCC GAT GGC GTG CCG GCA GCG GGC GTG CCG GCT TTT GGC
EBPP [K 3 F 3 ] (SEQ ID NO: 16)	GTT CCA GCG TTT GGC GTG CCA GCG AAA GGT GTT CCG GCG TTT GGGGTT CCC GCG AAA GGT GTG CCG GCC TTT GGT GTG CCG GCC AAA GGC
EBPP [D 3 F 3 ] (SEQ ID NO: 17)	GTT CCA GCG TTT GGC GTG CCA GCG GAT GGT GTT CCG GCG TTT GGGGTT CCC GCG GAT GGT GTG CCG GCC TTT GGT GTG CCG GCC GAT GGC
EBPP [H 3 A 3 I 1 ] (SEQ ID NO: 18)	GTG CCG GCG CAT GGA GTT CCT GCC GCC GGT GTT CCT GCG CAT GGTGTA CCG GCA ATT GGC GTT CCG GCA CAT GGT GTG CCG GCC GCC GGC
EBPP [H 5 G 1 ] (SEQ ID NO: 19)	GTT CCG GCC GGA GGT GTA CCG GCG CAT GGT GTT CCG GCA CAT GGTGTG CCG GCT CAC GGT GTG CCT GCG CAT GGC GTT CCT GCG CAT GGC
EBPP [G 1 C 3 F 2 ] (SEQ ID NO: 20)	GTG CCG GCG TGC GGC GTT CCA GCC TTT GGT GTG CCA GCG TGC GGA GTT CCG GCC GGT GGC GTG CCG GCA TGC GGC GTG CCG GCT TTT GGC
EBPPI [G 1 A 4 F 1 ] (SEQ ID NO: 21)	ATT CCT GCA GCC GGT ATC CCG GCC GGT GGC ATT CCG GCA GCC GGC ATT CCG GCC GCC GGC ATC CCG GCA TTT GGC ATT CCT GCA GCA GGC
EBPPI [G 1 A 3 F 2 ] (SEQ ID NO: 22)	ATT CCG GCC GCA GGC ATT CCT GCA TTT GGT ATT CCG GCG GCA GGC ATT CCT GCC GGT GGC ATC CCG GCA GCG GGC ATT CCG GCC TTT GGC

Amino acid sequence of the EBP library

EBPE [A 1 G 4 I 1 ] (SEQ ID NO: 23)	VPGGG	VPGAG	VPGGG	VPGGG	VPGIG	VPGGG
EBPP [A 1 G 4 I 1 ] (SEQ ID NO: 24)	VPAGG	VPAAG	VPAGG	VPAGG	VPAIG	VPAGG
EBPE [K 1 G 4 I 1 ] (SEQ ID NO: 25)	VPGGG	VPGKG	VPGGG	VPGGG	VPGIG	VPGGG
EBPP [K 1 G 4 I 1 ] (SEQ ID NO: 26)	VPAGG	VPAKG	VPAGG	VPAGG	VPAIG	VPAGG
EBPE [D 1 G 4 I 1 ] (SEQ ID NO: 27)	VPGGG	VPGDG	VPGGG	VPGGG	VPGIG	VPGGG
EBPP [D 1 G 4 I 1 ] (SEQ ID NO: 28)	VPAGG	VPADG	VPAGG	VPAGG	VPAIG	VPAGG
EBPE [E 1 G 4 I 1 ] (SEQ ID NO: 29)	VPGGG	VPGEG	VPGGG	VPGGG	VPGIG	VPGGG
EBPP [E 1 G 4 I 1 ] (SEQ ID NO: 30)	VPAGG	VPAEG	VPAGG	VPAGG	VPAIG	VPAGG
EBPE [G 1 A 3 F 2 ] (SEQ ID NO: 31)	VPGAG	VPGFG	VPGAG	VPGGG	VPGAG	VPGFG
EBPP [G 1 A 3 F 2 ] (SEQ ID NO: 32)	VPAAG	VPAFG	VPAAG	VPAGG	VPAAG	VPAFG
EBPP [K 1 A 3 F 2 ] (SEQ ID NO: 33)	VPAAG	VPAFG	VPAAG	VPAGG	VPAAG	VPAFG
EBPP [D 1 A 3 F 2 ] (SEQ ID NO: 34)	VPAAG	VPAFG	VPAAG	VPAGG	VPAAG	VPAFG
EBPP [K 3 F 3 ] (SEQ ID NO: 35)	VPAFG	VPAKG	VPAFG	VPAKG	VPAFG	VPAKG
EBPP [D 3 F 3 ] (SEQ ID NO: 36)	VPAFG	VPADG	VPAFG	VPADG	VPAFG	VPADG
EBPP [H 3 A 3 I 1 ] (SEQ ID NO: 37)	VPAHG	VPAAG	VPAHG	VPAIG	VPAHG	VPAAG
EBPP [H 5 G 1 ] (SEQ ID NO: 38)	VPAGG	VPAHG	VPAHG	VPAHG	VPAHG	VPAHG
EBPP [G 1 C 3 F 2 ] (SEQ ID NO: 39)	VPACG	VPAFG	VPACG	VPAGG	VPACG	VPAFG
EBPPI [G 1 A 4 F 1 ] (SEQ ID NO: 40)	IPAAG	IPAGG	IPAAG	IPAAG	IPAFG	IPAAG
EBPPI [G 1 A 3 F 2 ] (SEQ ID NO: 41)	IPAAG	IPAFG	IPAAG	IPAGG	IPAAG	IPAFG

In the present invention, the mussel triglyceride protein (MFP) may be characterized in that the mussel californianus foot protein 5 (Msselp5) or the mussel galloprovincialis foot protein 5 (Mgfp5). have.

In the present invention, MFP [Mgfp5] _n and MFP [Mcfp5] _n represents the type of mussel species and ligature proteins in the letters and numbers in parentheses, subscript 'n' indicates the number of repetitions of the MFP block. For example, MFP [Mgfp5] ₁ means Mediterranean mussel (mussel galloprovincialis), MFP type 5, and one MFP block repeat unit. Finally, double and triple block copolys consisting of EBP and MFP Peptides are represented by the construction of an MFP block and an EBP block with a hyphen between the two blocks, for example, a double block copeptide is an EBPPI [G1A4F1] _n -MFP [Mgfp5] _n, and a triple block copolypeptide is an EBPPI [ G1A4F1] _n -MFP [Mgfp5] _n -EBPPI [G1A4F1] _n .

Gene and amino acid sequences of the MFP block of the present invention are shown in Tables 3 and 4, respectively.

Gene Sequences in the MFP Library

MFP [Mgfp5] (SEQ ID NO: 42)	TCT AGT GAA GAA TAT AAA GGT GGT TAT TAC CCC GGC AAC ACC TAT CAT TAT CAT AGT GGG GGC AGT TAT CAC GGC AGC GGC TAC CAT GGC GGC TAT AAA GGT AAA TAC TAC GGT AAA GCG AAA AAA TAC ATC GGC AAA TAT AAG TAC CTG AAA AAA GCT CGT AAA TAC CAT CGT AAA GGC TAT AAA AAA TAT TAC GGC GGC GGC AGT TCG
MFP [Mcfp5] (SEQ ID NO: 43 )	GTG GGT AGC GGC TAT GAC GGC TAT TCA GAT GGC TAC TAT CCT GGT AGT GCA TAT AAC TAC CCG TCA GGG TCC CAT GGC TAC CAT GGT CAT GGC TAT AAA GGC AAA TAC TAT GGC AAA GGC AAA AAA TAT A C GGC AAG TAT AAA TAT CTG AAA AAA GCG CGC AAA TAT CAT CGC AAG GGC TAT AAA AAA TAC TAT GGT GGC GGC TCC AGT

Amino acid sequence of the MFP library

MFP [Mgfp5] (SEQ ID NO: 44)	SSEEYKGGYY PGNTYHYHSG GSYHGSGYHG GYKGKYYGKA KKYYYKYKNS GKYKYLKKAR KYHRKGYKKY YGGGSS
MFP [Mcfp5] (SEQ ID NO 45)	VGSGYDGYSD GYYPGSAYNY PSGSHGYHGH HYKGKYYGKG KKYYYKYKRT GKYKYLKKAR KYHRKGYKKY YGGGSS

In the present invention, the multi-block copolypeptide is characterized by the amino acid sequence of SEQ ID NO: 50 to 70, it may be characterized by the nucleotide sequence of SEQ ID NO: 71 to 91. In view, the present invention relates to a gene encoding the multiblock copolypeptide.

In another aspect, the present invention relates to a recombinant vector comprising the gene.

In another aspect, the present invention relates to a recombinant microorganism into which the gene or the recombinant vector is introduced.

In the present invention, the recombinant microorganism may be characterized in that the expression vector containing a gene encoding a tyrosinase (tyrosinase) or a gene encoding a tyrosinase is further introduced and co-expressed.

In the present invention, the mussel quartet protein (MFP) forms a waveguide having a catechol side chain in which tyrosine residues are hydroxylated by tyrosinase, and the waveguide is a metal ion or oxide through coordination bond or hydrogen bond. And semimetals.

In the present invention, the waveguide is specifically dyed in the NBT and glycinate solution due to the redox reaction.

In the present invention, orf438 gene may be further included in the expression vector.

In another aspect, the present invention provides a method for producing a multi-block copolypeptide by culturing the recombinant microorganism; And (b) obtaining the generated multiblock copolypeptide.

In the present invention, the recombinant microorganism of step (a), the expression vector containing a gene encoding a tyrosinase (tyrosinase) or a gene encoding tyrosinase is further introduced to the multi-block copolypeptide and tyro It may be characterized by the co-expression of cinases.

In the present invention, the tyrosine residue of the multi-block copolypeptide may be characterized in that the tyrosinase is modified into a dopa (3,4-dihydroxyphenylalanine) residue.

In the present invention, tyrosinase (tyrosinase) is expensive, according to the method for producing a multi-block copolypeptide of the present invention, it is economical because it can express a large amount of tyrosinase in bacteria.

In the present invention, the vector refers to a DNA preparation containing a nucleotide sequence of the polynucleotide encoding the target protein operably linked to a suitable control sequence to express the target protein in a suitable host cell. The regulatory sequence may comprise a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating the termination of transcription and translation, as desired It can be manufactured in various ways. The promoter of the vector may be constitutive or inducible. After being transformed into a suitable host, the vector can replicate or function independently of the host genome and integrate into the genome itself.

The vector used in the present invention is not particularly limited as long as it can be replicated in a host cell, and any vector known in the art may be used. Examples of commonly used vectors include natural or recombinant plasmids, phagemids, cosmids, viruses and bacteriophages. For example, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A can be used as a phage vector or cosmid vector, and pBR, pUC, pBluescriptII, pGEM system, pTZ system, pCL system and pET system can be used. The vector usable in the present invention is not particularly limited and known expression vectors can be used.

The expression “expression control sequence” refers to a DNA sequence that is essential for the expression of a coding sequence operably linked in a particular host organism. Such regulatory sequence is a promoter for transcription, for controlling such transcription. Any operator sequence, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence that controls termination of transcription and translation, for example, a regulatory sequence suitable for prokaryotes includes a promoter, optionally an operator sequence, and a ribosomal binding site Eukaryotic cells include promoters, polyadenylation signals, and enhancers, the most influential factors affecting the expression levels of genes in the plasmids, the promoters for high expression, the SRα promoter and cytomegalovirus. Derived promoters and the like are preferably used.

To express the DNA sequences of the invention, any of a wide variety of expression control sequences can be used in the vector. Examples of useful expression control sequences include, for example, early and late promoters of SV40 or adenovirus, lac system, trp system, TAC or TRC system, T3 and T7 promoters, major operator and promoter region of phage lambda, fd Regulatory regions of the code protein, promoters for 3-phosphoglycerate kinase or other glycolysis enzymes, promoters of the phosphatase such as Pho5, promoters of the yeast alpha-crossing system and prokaryotic or eukaryotic cells or viruses thereof And other sequences of constitution and induction known to modulate the expression of the genes, and various combinations thereof. The T7 RNA polymerase promoter Φ 10 may be usefully used to express protein NSP in E. coli.

Nucleic acids are "operably linked" when placed in a functional relationship with other nucleic acid sequences. This may be genes and regulatory sequence (s) linked in such a way as to allow gene expression when appropriate molecules (eg, transcriptional activating proteins) bind to regulatory sequence (s). For example, DNA for a pre-sequence or secretion leader is operably linked to DNA for a polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, "operably linked" means that the linked DNA sequence is in contact, and in the case of a secretory leader, is in contact and present within the reading frame. However, enhancers do not need to touch. Linking of these sequences is performed by ligation (linking) at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used.

As used herein, the term "expression vector" generally refers to a fragment of DNA that is generally double stranded as a recombinant carrier into which fragments of heterologous DNA have been inserted. Here, heterologous DNA refers to heterologous DNA, which is DNA not naturally found in host cells. Once expression vectors are within a host cell, they can replicate independently of the host chromosomal DNA and several copies of the vector and their inserted (heterologous) DNA can be produced.

As is well known in the art, to raise the expression level of a transfected gene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host. Preferably, the expression control sequence and the gene of interest are included in one expression vector including the bacterial selection marker and the replication origin. If the expression host is a eukaryotic cell, the expression vector must further comprise an expression marker useful in the eukaryotic expression host.

A wide variety of expression host / vector combinations can be used to express the genes encoding the polypeptides of the invention. Suitable expression vectors for eukaryotic hosts include, for example, expression control sequences derived from SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus and retrovirus. Expression vectors that can be used in bacterial hosts include a broader host range, such as bacterial plasmids, RP4, which can be exemplified in E. coli such as pBluescript, pGEX2T, pUC vectors, colE1, pCR1, pBR322, pMB9 and derivatives thereof. Phage plasmids, phage DNA that can be exemplified by a wide variety of phage lambda derivatives such as λgt10 and λgt11, NM989, and other DNA phages such as M13 and filamentary single-stranded DNA phages. Useful expression vectors for yeast cells are 2μ plasmids and derivatives thereof. A useful vector for insect cells is pVL 941.

Host cells transformed or transfected with the expression vectors described above constitute another aspect of the present invention. As used herein, the term “transformation” means introducing DNA into a host so that the DNA is replicable as an extrachromosomal factor or by chromosomal integration. As used herein, the term "transfection" means that the expression vector is accepted by the host cell whether or not any coding sequence is actually expressed.

The host cell of the invention is a recombinant microorganism into which a vector having a polynucleotide encoding at least one target protein is introduced, or a polynucleotide encoding at least one target protein is introduced into the microorganism so that the polynucleotide is integrated into a chromosome to express the target protein. Refers to a recombinant microorganism infected with a trait. It may be a prokaryotic or eukaryotic cell. In addition, a host having a high DNA introduction efficiency and a high expression efficiency of the introduced DNA is usually used. Known eukaryotic and prokaryotic hosts such as Escherichia coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such as Spodoptera pruperferda (SF9), animal cells such as CHO and mouse cells, COS 1, COS African green monkey cells such as 7, BSC 1, BSC 40 and BMT 10, and tissue cultured human cells are examples of host cells that can be used. In the case of using COS cells, since SV40 large T antigen is expressed in COS cells, the plasmid having the origin of replication of SV40 is present as a large number of copies of the episome in the cells. And expression can be expected. The introduced DNA sequence may be obtained from the same species as the host cell, may be of a different species than the host cell, or it may be a hybrid DNA sequence comprising any heterologous or homologous DNA.

Of course, it should be understood that not all vectors and expression control sequences function equally in expressing the DNA sequences of the present invention. Likewise not all hosts function equally for the same expression system. However, those skilled in the art can make appropriate choices among various vectors, expression control sequences and hosts without departing from the scope of the present invention without undue experimental burden. For example, in selecting a vector, the host must be considered, since the vector must be replicated in it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered. In selecting expression control sequences, several factors must be considered. For example, the relative strength of the sequence, the controllability, and the compatibility with the DNA sequences of the present invention should be considered, particularly with regard to possible secondary structures. Single cell hosts may be selected from a host for the selected vector, the toxicity of the product encoded by the DNA sequence of the invention, the secretory properties, the ability to accurately fold the protein, the culture and fermentation requirements, the product encoded by the DNA sequence of the invention from the host. It should be selected in consideration of factors such as the ease of purification. Within the scope of these variables, one skilled in the art can select a variety of vector / expression control sequence / host combinations capable of expressing the DNA sequences of the invention in fermentation or large scale animal culture. As a screening method when cloning the cDNA of the NSP protein by expression cloning, a binding method (binding method), a panning method (panning method), a film emulsion method (film emulsion method) and the like can be applied.

In the present invention, as a method of inserting the gene on the chromosome of the host cell, a conventionally known genetic manipulation method may be used, and the non-viral delivery method may be cell perforation, lipofection, microinjection, ballistic method, virosome, liposome. , Immunoliposomes, polyvalent cations or lipid: nucleic acid conjugates, naked DNA, artificial virons, and chemical promoted DNA influx. Sonorization, for example methods using the Sonitron 2000 system (Rich-Mar), can also be used for the delivery of nucleic acids. Other representative nucleic acid delivery systems are Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Maryland). And BTX Molesular Syetem (Holliston, Mass.). Lipofection methods are specified in US Pat. No. 5,049,386, US Pat. No. 4,946,787 and US Pat. No. 4,897,355 and lipofection reagents are commercially available, for example Transfectam ™ and Lipofectin ™. Suitable cations or neutral lipids for effective receptor-recognition lipofection of polynucleotides include lipids from Felgner (WO91 / 17424 and WO91 / 16024) and can be delivered to cells via in vitro introduction and to target tissues via in vivo introduction. have. Methods of preparing lipid: nucleic acid complexes, including target liposomes, such as immunolipid complexes, are well known in the art (Crystal, Science., 270: 404-410, 1995; Blaese et al., Cancer Gene Ther., 2: 291). 297, 1995; Behr et al., Bioconjugate Chem., 5: 382389, 1994; Remy et al., Bioconjugate Chem., 5: 647-654, 1994; Gao et al., Gene Therapy., 2: 710- 722, 1995; Ahmad et al., Cancer Res., 52: 4817-4820, 1992; US Patent 4,186,183; US Patent 4,217,344; US Patent 4,235,871; US Patent 4,261,975; US Patent 4,485,054 US Patent 4,501,728 US Patent 4,774,085 US Patent 4,837,028 US Patent 4,946,787.

The affinity of retroviruses can be altered by integrating with outer envelope proteins, thereby expanding the type of target cell. Lentiviral vectors are retroviral vectors that generate high viral titers by transducing or infecting non-dividing cells. The target tissue determines the retroviral gene persistence system. Retroviral vectors contain cis acting long terminal repeats that can pack 6-10 kb outer sequences. Minimal cis acting LTRs sufficient for replication and packaging of the vector can be used to integrate the therapeutic gene into target cells for permanent transgene expression. Widely used retroviral vectors include murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), monkey immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combination viruses thereof (Buchscher et al. , J. Virol., 66: 2731-2739, 1992; Johann et al., J. Virol., 66: 1635-1640 1992; Sommerfelt et al., Virol., 176: 58-59, 1990; Wilson et al. , J. Virol., 63: 2374-2378, 1989; Miller et al., J. Virol., 65: 2220-2224, 1991; PCT / US94 / 05700.

Transient expression of sucrose phosphorylase proteins is more common with adenovirus-based systems, and adenovirus-based vectors cause high efficiency transduction in many cells but do not require cell division. The vector allows for high titers and high levels of expression and can be produced in large quantities in a simple system. Adeno accessory virus (AAV) vectors are also used to transduce into cells with target nucleic acids, for example for the production of nucleic acids and peptides in vitro and for gene therapy in vivo and in vitro (West et al., Virology., 160: 38-47, 1987; US Pat. No. 4,797,368; WO93 / 24641; Kotin, Human Gene Therapy., 5: 793-801, 1994; Muzyczka, J. Clin. Invest., 94: 1351, 1994 The construction of recombinant AAV vectors is already known (US Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol., 5: 3251-3260, 1985; Tratschin, et al., Mol. Cell. Biol. , 4: 20722081, 1984; Hermonat & Muzyczka, PNAS., 81: 6466-6470, 1984; Samulski et al., J Virol., 63: 038223828, 1989). In clinical trials, gene transfer methods using at least six viral vectors are used, which complement the defective vector by inserting the gene into a helper cell line that generates transgenes. pLASN and MFG-S are examples of retroviruses used in clinical trials (Dunbar et al., Blood., 85: 3048-305, 1995; Kohn et al., Nat. Med., 1: 1017-102, 1995 Malech et al., PNAS., 94: (22) 12133-12138, 1997), PA317 / pLASN was the first therapeutic vector used in gene therapy (Blaese et al., Science., 270: 475-480, 1995) Transduction efficiency of MFG-S packaging vector was 50% or higher (Ellem et al., Immunol Immunother., 44 (1): 10-20, 1997; Dranoff et al., Hum. Gene Ther. , 1: 111-2, 1997).

Recombinant adeno-associated virus vectors (rAAV) are promising alternative gene delivery systems based on defective and nonpathogenic type 2 parvovirus adeno-associated viruses. All vectors are derived from plasmids with AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene delivery and stable transgene delivery due to integration into the genome of transduced cells is a great advantage of the vector system (Wagner et al., Lancet., 351: 9117-17023, 1998; Kearns et al., Gene Ther., 9: 748-55, 1996).

In the present invention, "co-expression" means that two or more genes are expressed at the same time, in the present invention is expressed as co-expression.

In one embodiment of the invention, tyrosine residues of the MFP block were hydroxylated in two ways ((1) bacterial coexpression system (orf438 and tyrosinase and block copolypeptides co-expressed in E. coli) and (2 ) Mushroom-derived tyrosinase catalysis). Double and triple block copeptides composed of EBP (A block) and MFP (B block) are designed in AB-, ABA- and BAB-types to form self-assembled micellar structures and injectable hydrogels, It can be used as a bio coating and a bioadhesive material having. First, tyrosine residues of the purified EBP-MFP block copolypeptides were transformed to DOPA by mushroom derived tyrosinase. To investigate surface adhesion, each block copolypeptide was dissolved in 10 mM phosphate buffer (pH 5) (to prevent DOPA self-oxidation) and incubated with the oxidant NaIO ₄ (for cross-molecular crosslinking). . As a result, depending on the oxidant concentration and the block copolypeptide type in the range of 10 mM to 100 mM, different surface adhesive strengths were shown. Through this, it can be seen that DOPA and quinone are essential elements exhibiting bulk adhesiveness. Dopa (DOPA) in MFP plays an important role in surface adhesion, and quinone, an oxidized form of DOPA, imparts cohesion through intermolecular crosslinking. In addition, EBP blocks with phase transitions above LCST exhibit improved cohesion for physically crosslinked hydrogelation and surface adhesion. Second, the EBP-MFP block copolypeptide and orf438 and tyrosinase were co-expressed in E. coli to hydroxylate the tyrosine residues of the block copolypeptides without further treatment. As a result, it was confirmed that the EBP-MFP block copolypeptide has superior adhesion compared to the EBP-MFP block copolypeptide modified by mushroom-derived tyrosinase, it can be seen that it is useful for industrial scale up (Fig. 12). In the present invention, the surface adhesion strength of the block copolypeptides co-expressed in the form of micelles and hydrogels was investigated. As a result, it was confirmed that the EBP-MFP block copolypeptide of the present invention has great potential as micelles and hydrogels having surface adhesion (Figs. 10, 11, 13 and 14).

In another aspect, the present invention relates to a self-assembled nanostructure of a core-shell structure in which the multiblock copolypeptide is a temperature stimulus so that the EBP block forms a core structure and the MFP block forms a shell structure.

In the present invention, the core-shell structure refers to a micelle structure, and micelles generally refer to a thermodynamically stable and uniform spherical structure formed by low molecular weight materials having both amphiphilic, for example, hydrophilic and hydrophobic groups. . When a non-aqueous drug is dissolved and added to the compound having the micelle structure, the drug is present in the micelle, and the micelle can release the target-oriented drug in response to a change in temperature or pH in the body, thereby serving as a carrier for drug delivery. The possibility of application is very high.

In the present invention, MFPs with different block lengths were fused with EBPPI to form self-assembled nanostructures by thermal stimulation. Molecules of the EBPPI-MFP biblock copolypeptide can self assemble into core-shell nanostructures in response to temperature (FIG. 1 (A)). MFP is fused to the N-terminus or C-terminus of EBPPI [G1A4F1] ₆ to exhibit surface adhesion under moisture conditions. Surface adherent micelles can be applied to the surface-coated nanostructures on the stent, which can be inserted into the lumen of anatomical vessels to maintain passage and drug delivery carriers.

In another aspect, the present invention relates to a drug delivery composition comprising the self-assembled nanostructure.

The self-assembled nanostructures according to the present invention can be used as an extracellular matrix as an effective scaffold for drug delivery. The drug is not particularly limited and includes chemicals, small molecules, peptide or protein medicines, nucleic acids, viruses, antibacterial agents, anticancer agents, anti-inflammatory agents and the like.

The small molecules may be, for example, but not limited to, contrast agents (eg, T1 contrast agents, T2 contrast agents such as superparamagnetics, radioisotopes, etc.), fluorescent markers, dyeing materials, and the like.

The peptide or protein drug product may be a hormone, a hormone analog, an enzyme, an enzyme inhibitor, a signaling protein or a part thereof, an antibody or a part thereof, a single chain antibody, a binding protein or a binding domain, an antigen, an adhesion protein, a structural protein, a regulatory protein, a toxin Proteins, cytokines, transcriptional regulators, blood clotting factors, vaccines, and the like. More specifically, fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), transforming growth factor (TGF), bone morphogenetic protein BMP), human growth hormone (hGH), pig growth hormone (pGH), leukocyte growth factor (G-CSF), red blood cell growth factor (EPO), macrophage growth factor (M-CSF), tumor necrosis factor (TNF) , Epidermal growth factor (EGF), platelet-induced growth factor (PDGF), interferon, interleukin, calcitonin, nerve growth factor (NGF), growth hormone releasing factor, engiotensin, luteinizing hormone releasing hormone (LHRH), corpus luteum LHRH agonist, insulin, thyroid-stimulating hormone releasing hormone (TRH), endostatin, endostatin, somatostatin, glucagon, endorphins, vacitracin, mergain, colistin, single antibodies, vaccines or these Containing a mixture of , But it is not limited thereto.

The nucleic acid can be, for example, RNA, DNA or cDNA, and the sequence of nucleic acids can be a coding site sequence or a non-coding site sequence (eg antisense oligonucleotide or siRNA).

The virus may be a virus core (ie, a nucleic acid of a virus packaged without a virus envelope) comprising the virus or the nucleic acid of the virus. Examples of viruses and viral cores that can be transported include, but are not limited to, papilloma virus, adenovirus, baculovirus, retroviral core and semilky virus core.

The antimicrobial agent may be minocycline, tetracycline, oploxacin, phosphomycin, mergaine, profloxacin, ampicillin, penicillin, doxycycline, thienamycin, cephalosporin, norcardycin, gentamicin, neomycin, kanamycin , Paromomycin, micronomycin, amikacin, tobramycin, dibecasin, cytotaxin, cefacller, erythromycin, ciprofloxacin, levofloxacin, endoxacin, vancomycin, imipenem, fusidic acid and mixtures thereof It may be, but is not limited thereto.

The anticancer agents include paclitaxel, taxotier, adriamycin, endostatin, angiostatin, mitomycin, bleomycin, cisplatin, carboplatin, doxorubicin, daunorubicin, idarubicin, 5-fluorouracil, methotrexate, exec Tinomycin-D and mixtures thereof, but is not limited thereto.

The anti-inflammatory agents are acetaminophen, aspirin, ibuprofen, diclofenac, indomethacin, pyricampam, phenopropene, flubiprofen, ketoprofen, naproxen, suprofen, roxofene, synoxycamp , Tenoxycam, and mixtures thereof, but is not limited thereto.

In another aspect, the present invention relates to a hydrogel in which the multiblock copolypeptide is prepared by forming crosslinks between block polypeptides by temperature stimulation.

Hydrogel of the present invention has a mechanical flexibility similar to the actual tissue, and contains a lot of water, but since the bond of the gel is not broken by water, it requires adhesion with a wet biological surface containing water and external moisture Applications to medical adhesives and the like that must have resistance to Edo have been actively made. Accordingly, the hydrogel having excellent tissue adhesion according to the present invention is capable of various biomedical applications such as tissue adhesives or hemostatic agents, tissue engineering supports, drug delivery carriers, tissue fillers, wound healing, and intestinal adhesion prevention.

In the present invention, two triple block copolypeptides of various block order and length were constructed. MFP [Mgfp5] ₁ -EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ and EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ -EBPPI [G ₁ A ₄ F ₁ ] ₆ Triple block copolypeptides were prepared as injectable hydrogels with surface adhesion (FIGS. 1B and 1C).

In the present invention, the EBP block is used as a physical crosslinker with stimulatory reactivity, while the MFP block is used for the introduction of chemical crosslinking and surface adhesion by quinone formation. Triple block copolypeptides, including MFP-EBP-MFP and EBP-MFP-EBP, can be self-assembled into hydrogels by oxidation by temperature change and NaIO ₄ treatment. 1 (D) shows the mechanism of surface adhesion, intermolecular crosslinking and stimulus reactivity of the triple block copolypeptides.

In the present invention, the hydrogel may be formed by the oxidative or non-covalent interaction of the dopa (3,4-dihydroxyphenylalanine) residue contained in the mussel musa protein (MFP).

In another aspect, the present invention relates to a bioadhesive composition comprising the hydrogel.

The bioadhesive composition of the present invention can be used in various areas such as skin, blood vessels, digestive organs, cranial nerves, plastic surgery, orthopedics, and the like, by replacing cyanoacrylic adhesives or fibrin adhesives that are mainly used in the market. For example, the biocompatible biotissue adhesive of the present invention can replace surgical sutures, can be used to block unnecessary blood vessels, and can be used for hemostasis and suturing of soft tissues such as facial tissues and cartilage, and hard tissues such as bones and teeth. It is possible to apply as a home medicine. 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 may be applied to the internal and external surfaces of the human body, that is, the bioadhesive of the present invention may be applied to the external surface of the human body such as skin or the surface of an internal organ exposed during a surgical procedure. Can be applied as In addition, the bioadhesives of the present invention can be used to bond damaged parts of a tissue, to seal leakage of air / fluid from the tissue, to adhere a medical device to the tissue, or to fill a defective portion of the tissue. The term “biological tissue” herein is not particularly limited and includes, for example, skin, bones, nerves, axons, cartilage, blood vessels, corneas, muscles, fascia, brain, prostate, breast, endometrium, lung, spleen, small intestine. , Liver, testes, ovaries, cervix, rectum, stomach, lymph nodes, bone marrow and kidneys.

In another embodiment, the bioadhesive of the present invention may 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 closure. That is, the bioadhesive of the present invention may be applied topically and used to seal the wound, replacing the suture. In addition, the bioadhesive of the present invention can be applied to restoring hernia, for example, can be used for the surface coating of the mesh used for restoring hernia.

In another embodiment, the bioadhesive of the present invention can also be used to prevent closure and leakage of tubular structures such as blood vessels. In addition, the bioadhesive of the present invention can also be used for hemostasis.

In another embodiment, the bioadhesive of the present invention may be used as an anti-adhesion agent after surgery. Adhesion occurs at all surgical sites, where other tissues stick around the wound around the surgical site. Adhesion occurs 97% after surgery, especially 5-7% of which causes serious problems. To prevent these adhesions, the wound may be minimized during surgery or anti-inflammatory agents may be used. It also activates tissue plasminogen activators (TPAs) or physical barriers such as crystalline solutions, polymer solutions, and solid membranes to prevent fibrin formation, but these methods can be toxic in vivo and have other side effects. have. The bioadhesive of the present invention can be applied to exposed tissue after surgery to be used to prevent adhesions occurring between the tissue and surrounding tissue.

In another aspect, the present invention relates to a surgical suture comprising the hydrogel.

As another aspect, it may be related to a support for tissue engineering comprising a hydrogel of the present invention.

Tissue engineering technology refers to a method of culturing cells isolated from a patient's tissue in a support to prepare a cell-support complex, and then transplanting the prepared cell-support complex back into the human body. It is applied to the regeneration of almost all organs of the human body such as cartilage, artificial cornea, artificial blood vessel, artificial muscle. Bioadhesive hydrogels of the present invention can provide scaffolds similar to living tissue to optimize the regeneration of living tissue and organs in tissue engineering techniques. In addition, by using the support of the present invention can easily implement an artificial extracellular matrix, it can be used as a medical material such as cosmetics, wound dressings, dental matrix.

Hydrogels of the present invention can be easily attached to various bioactive substances involved in the action of promoting the growth and differentiation of cells through the interaction with the cells or tissues of the human body and help the regeneration and recovery of tissues. In addition, the physiologically active substance may collectively refer to various biomolecules that may be included to implement an artificial extracellular matrix having a structure similar to a natural extracellular matrix. Bioactive substances include cells, proteins, nucleic acids, sugars, enzymes, and the like, and examples thereof include cells, proteins, polypeptides, polysaccharides, monosaccharides, oligosaccharides, fatty acids, nucleic acids, and the like. have. The cell may be any cell including prokaryotic and eukaryotic cells. For example, osteoblast, fibroblast, hepatocyte, neurons, cancer cell, B cell, Immune cells and embryonic cells, including white blood cells, and the like. In addition, the physiologically active substance includes, but is not limited to, plasmid nucleic acid as a nucleic acid material, hyaluronic acid as a sugar substance, heparin sulfate, chondroitin sulfate, alginate, and hormonal protein as a protein substance.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Experimental Material Preparation

PET-21a vector and BL21 (DE3) E. coli cells were purchased from Novagen Inc. (Madison, WI, U.S.). Top 10 competent cells were purchased from Invitrogen (Carlsbad, CA, U.S.). Oligonucleotides were chemically synthesized by Cosmo Gene Tech (Seoul, South Korea). Fermentas (Ontario, Canada) was purchased from Fast AP, a thermosensitive alkaline phosphatase, and restriction endonucleases including BamHI and XbaI. Other restriction endonucleases were obtained from New England Biolabs (Ipswich, MA, U.S.), including BseRI, AcuI, and other restriction enzymes. T4 DNA ligase was obtained from Elpis Bio-tech (Taejeon, South Korea). All kits for DNA mini-preparation, gel extraction, and PCR purification were obtained from Geneall Biotechnology (Seoul, South Korea). Dyne Agarose High was obtained from DYNE BIO (Seongnam, South Korea). All Top10 cells were grown in TB DRY medium (MO BIO Laboratories, Carlsbad, CA, U.S.) and super optimal broth with catabolite repression (SOC) medium supplemented with 20 mM glucose (Formedium, UK). All BL21 (DE3) cells were grown in circular growth medium obtained from MP Biomedicals (Solon, OH, U.S.). Ready Gel (Tris-HCI, 2-20%), a precast gel, was obtained from Bio-Rad (Hercules, CA, U.S.). Phosphate buffered saline (PBS, pH 7.4), ampicillin and polyethyleneimine (PEI) were purchased from Sigma-Aldrich (St Louis, MO).

Example 2: Gene Synthesis of MFP

MFP gene was obtained from M. galloprovincialis- and M. californianus-foot protein 5. The pUC plasmid containing the MFP gene sequence was treated with a buffer containing 10 U of XbaI and 15 U of Acu1 at 37 ° C. for 30 to 60 minutes, and the mpET-21a plasmid vector was also subjected to restriction enzymes of 10 U of XbaI and 15 U of BseRI. Treated. Thereafter, 90 pmol of MFP dsDNA and 30 pmol of linearized mpET-21a cloning vector were incubated in T4 DNA ligase buffer containing 1 U of T4 DNA ligase for 30 minutes at 16 ° C. for ligation. The ligated plasmids were transformed into Top 10 chemical receptor cells and then applied onto SOC plates supplemented with 50 μg / ml ampicillin. The inserted sequence was then confirmed by DNA sequencing (Table 3).

To achieve strong adhesion with increased DOPA molecule counts, the gene sequence of MFP was multiplied up to four repeat units. Gene sequences and sizes encoding multimerized MFP were confirmed using DNA sequencing and DNA agarose gel electrophoresis.

As a result, as shown in Figure 2, it was confirmed that the MFP length appears to vary from 231bp to 924bp, because the MFP was repeated 1, 2 and 4 times.

Example 3: Gene Construction of Block Copolypeptides Consisting of EBP and MFP

Block copolypeptide libraries consisting of EBP and MFP were synthesized using plasmids with EBP or MFP single block genes. The plasmid containing EBP represented by the gene and amino acid sequence shown in Table 1 and Table 2 was treated with a buffer containing 10 U of XbaI and 15 U of BseRI for 30-60 minutes at 37 ° C., followed by PCR purification kit. Purification with Plasmids with MFP block gene were treated with a buffer containing 10 U of XbaI and 15 U of AcuI for 30-60 minutes at 37 ° C. The MFP gene represented by the amino acid sequence of Table 3 was isolated using agarose gel electrophoresis and purified by gel purification kit. Plasmids with EBP blocks were used as vectors and fused with the MFP gene as insert. Ligation was performed by incubating 90 pmol of purified insert and 30 pmol of linearized vector in ligase buffer containing 1 U of T4 DNA ligase for 30-60 minutes at 16 ° C. The product was then transformed into Top10 recipient cells and then streaked onto SOC plates supplemented with 50 μg / ml ampicillin. In order to synthesize double and triple block copolypeptides of EBP and MFP, the double block gene of EBPPI-MFP was synthesized by inserting the MFP gene at the 5 'or 3' end of the EBPPI gene. The MFP-EBPPI-MFP or EBPPI-MFP-EBPPI triple block gene was synthesized by inserting the MFP or EBPPI gene into the 5 'or 3' end of the previously synthesized EBPPI-MFP double block gene. Other double and triple block copolypeptide genes were synthesized by varying the block order and length of EBP and MFP.

Triple block copolypeptides were synthesized by a recursive directional ligation (RDL) method using double block copolypeptides as building blocks (FIG. 3 (B)). The EBPPI [G1A4F1] 6 gene was seamlessly fused to the N-terminus of the MFP [Mgfp5] 1-EBPPI [G1A4F1] 6 double block copolypeptide gene by RDL, resulting in EBPPI [G1A4F1] 6-MFP [Mgfp5] 1-EBPPI [G1A4F1] 6 triple block copolypeptide genes were synthesized.

The length and molecular weight of the double block copolypeptides are shown in Table 5 below.

Di-block copolypeptides	Nucleotide chain length (bp)	M.W (kDa)
MFP [Mgfp5] 1 -EBPP [G 1 A 3 F 2 ] 24 (SEQ ID NO: 50)	2400	69.55
MFP [Mgfp5] 2 -EBPP [G 1 A 3 F 2 ] 24 (SEQ ID NO: 51)	2631	78.47
MFP [Mgfp5] 4 -EBPP [G 1 A 3 F 2 ] 24 (SEQ ID NO: 52)	3093	96.31
MFP [Mgfp5] 1 -EBPPI [G 1 A 3 F 2 ] 6 (SEQ ID NO: 53)	780	25.23
MFP [Mgfp5] 2 -EBPPI [G 1 A 3 F 2 ] 6 (SEQ ID NO: 54)	1011	34.15
MFP [Mgfp5] 4 -EBPPI [G 1 A 3 F 2 ] 6 (SEQ ID NO: 55)	1470	51.98
MFP [Mgfp5] 1 -EBPPI [G 1 A 4 F 1 ] 6 (SEQ ID NO: 56)	780	24.40
MFP [Mgfp5] 2 -EBPPI [G 1 A 4 F 1 ] 6 (SEQ ID NO: 57)	1011	33.20
MFP [Mgfp5] 4 -EBPPI [G 1 A 4 F 1 ] 6 (SEQ ID NO: 58)	1470	50.15

The length and molecular weight of the triple block copolypeptides are shown in Table 6 below.

Tri-block copolypeptides	Chain length (bp)	M.W (kDa)
EBPPI [G 1 A 3 F 2 ] 6 -MFP [Mgfp5] 1 -EBPPI [G 1 A 3 F 2 ] 6 (SEQ ID NO: 59)	1320	41.13
EBPPI [G 1 A 3 F 2 ] 6 -MFP [Mgfp5] 2 -EBPPI [G 1 A 3 F 2 ] 6 (SEQ ID NO: 60)	1551	50.05
EBPPI [G 1 A 3 F 2 ] 6 -MFP [Mgfp5] 4 -EBPPI [G 1 A 3 F 2 ] 6 (SEQ ID NO: 61)	2010	67.88
EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 -EBPPI [G 1 A 4 F 1 ] 6 (SEQ ID NO: 62)	1320	40.10
EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 2 -EBPPI [G 1 A 4 F 1 ] 6 (SEQ ID NO: 63)	1551	49.50
EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 4 -EBPPI [G 1 A 4 F 1 ] 6 (SEQ ID NO: 64)	2010	67.10
MFP [Mgfp5] 1 -EBPPI [G 1 A 3 F 2 ] 6 -MFP [Mgfp5] 1 (SEQ ID NO: 65)	1050	34.16
MFP [Mgfp5] 2 -EBPPI [G 1 A 3 F 2 ] 6 -MFP [Mgfp5] 2 (SEQ ID NO: 66)	1500	52.40
MFP [Mgfp5] 4 -EBPPI [G 1 A 3 F 2 ] 6 -MFP [Mgfp5] 4 (SEQ ID NO: 67)	2436	94.77
MFP [Mgfp5] 1 -EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 1 (SEQ ID NO: 68)	1050	33.40
MFP [Mgfp5] 2 -EBPPI [G 1 A 4 F 1 ] 6 MFP [Mgfp5] 2 (SEQ ID NO: 69)	1500	52.35
MFP [Mgfp5] 4 -EBPPI [G 1 A 4 F 1 ] 6 -MFP [Mgfp5] 4 (SEQ ID NO: 70)	2436	88.03

Example 4: PCR and vector construction of tyrosinase and orf438 for bacterial co-expression

E. coli cells were grown in TB dry medium containing 50 μg / mL ampicillin. pIJ702, a plasmid containing tyrosinase and S. lividans comprising orf438 were obtained from the American Type Culture Collection (ATCC, 35387). Single colonies of S. lividans were grown at 30 ° C. in R2 YE medium. Plasmids containing both tyrosinase and orf438 were purified from S.lividans. The tyrosinase gene was amplified by polymerase chain reaction (PCR) using primers of pSA-tyr-5p and pSA-tyr-3 '(pSA-tyr-5p (SEQ ID NO: 46): 5′-g gaG GAT CCg acc gtc cgc aag aac cag-3 ′ and pSA-tyr-3 ′ (SEQ ID NO: 47): 5 ′ gga AAG CTT gac gtc gaa ggt gta gtg ccg −3 ′. Amplified PCR products were treated with BamI and HindIII. Similarly, the orf438 gene was amplified by PCR using primers of pSA-438-5 'and pSA-438-3' and the amplified product was treated with EcoRV and KpnI (pSA-438-5 '(SEQ ID NO: 48). ): 5'- c acG ATA TCg ccg gaa ctc acc cgt cgt-3 ', pSA-438-3' (SEQ ID NO: 49): 5'- caa GTT ACC gtt gga ggg gaa ggg gag gag-3 '. The expression vector pACYCDuet-1 plasmid (Merck, Darmstadt, Germany) was also subjected to the same restriction enzyme as the PCR product, and then the cleaved product was introduced. Finally, the DNA sequence was confirmed by DNA sequencing.

Example 5: Co-Expression and Block Copolypeptide Purification of Block Copolypeptides, orf438 and Tyrosinase

E. coli BL21 (DE3) cells containing pET21a with block copolypeptides and plasmids with orf438 and pACYC duet of tyrosinase were grown in Circlegrow medium. Colonies were inoculated in 50 mL TB medium supplemented with 50 μg / ml Ampicillin (Duchefa) and 50 μg / ml Chloramphenicol (Duchefa) per ml. The preculture was shaken overnight at 37 ° C. and 200 rpm. The precultured medium was inoculated with 500 mL of high nutrition medium (circlegrow) containing 50 μg / mL of ampicillin and chloramphenicol, and cultured at 37 ° C. and 200 rpm until the OD600 reached 0.6 to 0.8. To induce protein expression, Isopropyl- at a final concentration of 1 mM

-D-thiogalactopyranoside (IPTG) was added and further incubated overnight at 37 ° C and 200 rpm. Thereafter, cells were obtained by centrifugation at 4 ° C and 4,500 rpm for 10 minutes. The expressed EBPPI-MFP block copolypeptides were purified using inverse transition cycling (ITC). Cell pellets were resuspended in 5% acetic acid containing 8M urea. After sonication (VC-505, Sonic and materials Inc, Danbury, CT) for 10 seconds in an ice bath, cells were destroyed by 30 seconds of cooling (fooling). Cell lysates were centrifuged at 50 ° C. and 13000 rpm for 15 minutes in a 50 mL centrifuge tube to precipitate insoluble debris of the cell lysates. Subsequently, the supernatant containing the water-soluble EBPPI-MFP block copolypeptide was transferred to a new 50 mL centrifuge tube and centrifuged at 4 ° C. and 13000 rpm for 15 minutes to precipitate nucleic acid contaminants. Inverse transition cycling (ITC) of EBPPI was performed several times through the addition of sodium chloride at a final concentration of 0.5-1.0 M. EBPPI-MFP block copolypeptides were aggregated by the salt effect and separated from lysates by centrifugation for 15 minutes at 37 ° C., 13,000 rpm. Aggregated block copolypeptides were resuspended in wells containing 30 mL of sodium acetate buffer (pH 5.0) and 4M urea at 4 ° C. To remove any aggregated protein contaminants, the resuspended protein solution was centrifuged at 4 ° C. and 13,000 rpm for 15 minutes. The aggregation and resuspension treatment was repeated 5 to 10 times until the purity of the block copolypeptide reached about 95%. The purity was measured using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

For the hydroxylation of tyrosine residues of block copolypeptides, block copolypeptides, tyrosinase (˜32 kDa) and orf438 (˜15 kDa) were co-expressed in soluble form.

As a result, the recombinant EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptide was prepared from a pET21a vector comprising a double block copolypeptide and a pACYC duet vector comprising tyrosinase and orf438. Co-expressed with tyrosinase and orf438 in E. coli via a dual vector system.

As shown in FIG. 5 and FIG. 6 (A), tyrosinase and orf438 genes were identified in the amplified tyrosinase and orf438 encoded pACYC duet vectors.

Figure 6 (B) shows a double block copolypeptide of (1) bacterial expression of pET21 alone, (2) tyrosinase and orf438 of bacterial expression of pACYC duet alone, and (3) a coexpressed pET21a vector. Tyrosinase and orf438 of copolypeptide and pACYC duet vectors. According to the plasmid copy number of each vector, the block copolypeptide in the pET vector has a copy number (-40) that is higher than the copy number (-12) of the tyrosinase and the pACYC duet vector encoding orf438. That is, the block copolypeptide of the pET vector alone was expressed more than the coexpression system.

6 (C) also shows (1) a triple block copolypeptide of EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ -EBPPI [G ₁ A ₄ F ₁ ] ₆ alone with pET21, and (2) Tyrosinase and orf438 of the triple-block copolypeptide of pET21a vector and pACYC duet vector were identified.

As a result, as shown in FIGS. 6 (E) and 6 (F), the block copolypeptides co-expressed with tyrosinase and orf438 were stained purple, and the block copolypeptides expressed alone as controls were yellow. Stained with. Thus, it can be seen that hydroxylation occurs in the block copolypeptide by co-expression with tyrosinase, orf438.

Example 6: Hydroxylation of EBP-MFP Block Copolypeptide Tyrosine Residues by Mushroom-derived Tyrosinase

Tyrosine residues of the EBP-MFP block copolypeptide were converted to DOPA via modification by mushroom derived tyrosinase (Sigma Aldrich, T3824) (FIG. 6 (D)).

EBP-MFP block copolypeptide was resuspended in 10 mM phosphate buffer supplemented with 10 mM sodium borate and the pH was adjusted to pH 7.0 using ascorbic acid. Then, mushroom-derived tyrosinase at a final concentration of 0.01 mg / ml was added. The solution was gently incubated for 3 hours at room temperature (RT). The tyrosinase-treated EBP-MFP block copolypeptides were phase transitioned at 40 ° C as the temperature increased, and purified by centrifugation at 16,000 rpm for 10 minutes at 40 ° C to remove tyrosinase. It was. Aggregated modified block copolypeptides were resuspended in 10 mM phosphate (pH 5) in an ice bath and the sample was centrifuged at 16,000 rpm for 15 minutes to remove remaining insoluble material.

In particular, to prevent autooxidation during the purification step, EBP-MFP block copolypeptide was added as a 5% acetic acid (pH 3) solution and lyophilized. This is because quinone, the oxidized form of DOPA, induces intermolecular covalent bonds and reduces interaction with surface molecules in oxidizing conditions.

EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptides expressed alone in E. coli were purified by ITC, and tyrosine residues of the double block copolypeptides were mushroom-derived tyrosinase-. It was transformed into DOPA by catalytic reaction.

As shown in FIG. 7, the hydroxylated double block copolypeptides developed purple in NBT staining due to the hydroxylation of tyrosine residues. In contrast, unmodified double block copolypeptides developed as whitish yellow as controls.

Example 7: Characterization of EBP-MFP Block Copolypeptides

The purity and molecular weight of all block copolypeptides were characterized by SDS-PAGE including copper staining. The phase transition behavior of the block copolypeptides was characterized by UV-visible spectrophotometer.

MFPs with different block lengths were fused with EBPPI to form self-assembled nanostructures by thermal stimulation.

EBPPI-MFP double block copolypeptides, MFP-EBPPI-MFP triple block copolypeptides, and EBPPI-MFP-EBPPI triple block copolypeptides with concentrations above 12.5 uM have a hydrodynamic radius (R _h ) of 20 nm to 40 nm. Eggplants self-assemble into core-shell nanostructures, but the triple block copolypeptides under concentrated conditions formed hydrogels in response to temperature.

8 (A) and 8 (B) show mushroom derived tyrosinase-catalyzed EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptide and mushroom derived tyrosinase-catalyzed, respectively. EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ -EBPPI [G ₁ A ₄ F ₁ ] ₆ Triple block copolypeptides show copper stained SDS-PAGE photographic images. The MW of the hydroxylated EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptides is similar to the expected MW (~ 24.6 kDa) and the hydroxylated EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ -EBPPI [G ₁ A ₄ F ₁ ] _{6 The} triple block copolypeptides are also similar to the expected MW (~ 40.1 kDa).

Multimerization forms of hydroxylated EBPPI [G1A4F1] 6-MFP [Mgfp5] 1 double block copolypeptides of 48.0 kDa and 92.0 kDa were identified.

Likewise, hydroxylated EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ -EBPPI [G ₁ A ₄ F ₁ ] ₆ triple block copolypeptides are multiplexed to ˜80.0 kDa and ˜120.0 kDa It was confirmed.

 Because of the intermolecular crosslinking caused by the quinone formed by autooxidation of DOPA, multiplexed forms of hydroxylated double- and triple-block copolypeptides were formed in 10 mM phosphate buffer (pH5.0). This means that mushroom derived tyrosinase-catalyzed hydroxylation of the block copolypeptide has occurred.

FIG. 8 (C) shows EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptides treated with various concentrations of oxidizer of NaIO ₄ and copper stained SDS in their hydroxylated form. -PAGE image. Treatment with NaIO _{4 results} in intermolecular crosslinking of unmodified EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptides due to the formation of di tyrosine residues, resulting in SDS The chemically crosslinked double block copolypeptides remain in the wells during PAGE (lanes (2-3) in FIG. 8 (C)).

On the other hand, hydroxylated double block copolypeptides were multiplexed due to the formation of di tyrosine residues at neutral pH as well as the formation of quinones induced by DOPA self oxidation. That is, NaIO ₄ induced intermolecular crosslinking through the formation of quinone and di tyrosine residues, and no migration of chemically crosslinked hydroxylated double block copolypeptides occurred during SDS-PAGE.

The thermal properties of EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptides range from 10 ° C. to 1 ° C./min heating rate in 10 mM phosphate buffer (pH 5, to prevent oxidation). It observed by measuring 350 nm absorbance in the temperature range of 70 degreeC.

As a result, FIG. 9 (A) shows EBPPI [G ₁ A ₄ F ₁ ] ₆ , 25 μM of mushroom-derived tyrosinase-catalyzed hydroxylation with or without the control EBPPI [G ₁ A ₄ F ₁ ] Thermal profile of _6- MFP [Mgfp5] ₁ double block copolypeptide. Monoblock, EBPPI [G ₁ A ₄ F ₁ ] ₆ exhibited complete solubility under T _t (approx. 45 ° C.), aqua conditions, and abruptly above LCST due to total aggregation of EBPPI [G ₁ A ₄ F ₁ ] ₆ Metastasis was shown.

In contrast, EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptides exhibit different thermal reactivity from the monoblock of EBPPI. Since the MFP block makes EBPPI [G ₁ A ₄ F ₁ ] ₆ more hydrophobic, the LCST of EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptides was induced by mushroom tyrosinase. It decreased to 35 ° C. regardless of hydroxylation. In addition, the absorbance of EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptides above the transition temperature shows thermally-induced aggregation of the EBPPI block (core) and water soluble MFP block (shell). , Self-assembling nanostructures were formed.

LCST behavior was analyzed according to the concentration of block copolypeptide and NaIO ₄ oxidant. As a result, 250 μM of the hydroxylated EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptide and 10 mM NaIO ₄ treated 25 μM double block copolypeptide exhibited rapid transition of EBP block. 9 (B) and 9 (C). This is due to the formation of quinones induced by DOPA self oxidation and non-covalent interactions of the MFP blocks as well as di tyrosine residues at neutral pH. In particular, the aggregation of high concentrations of MFP block is hydrogen bond,

Induced by non-covalent interactions such as stacking, electrostatic and hydrophobic interactions.

9 (D) shows EBPPI [G ₁ A _{4 with} mushroom-derived tyrosinase-catalyzed hydroxylation of 25 μM measured 350 nm absorbance at 1 ° C./min in 10 mM phosphate buffer (pH 5). F ₁ ] ₆ -MFP [Mgfp5] ₁ Double Block Copolypeptide and EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ -EBPPI [G ₁ A ₄ F ₁ ] ₆ Triple Block Copolypeptides Characteristics. The LCST of the triple block decreased to 25 ° C. This result is 10 ° C. lower than EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptides. This is because hydrophobic EBPPI blocks were introduced at both ends of the MFP [Mgfp5] ₁ intermediate block. In addition, the thermal reactivity of the triple block copolypeptides is similar to the EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptides hydroxylated with 10 mM NaIO ₄ as shown in FIG. 9 (C). Do. Triple block copolypeptides aggregated at temperatures higher than the LCST of the EBPPI block. This is because EBPPI blocks in double and triple block copolypeptides can form both chemical crosslinking of MFP through physical crosslinking and quinone formation.

Example 8: Bulk scale surface adhesion analysis of (1) a block copolypeptide treated with mushroom derived tyrosinase and (2) a hydroxylated block copolypeptide in a co-expression system

In order to perform bulk scale shear strength tests on aluminum deposits, surface adhesion strengths were analyzed according to the concentration of block copolypeptides and NaIO ₄ .

Hydroxylated double block and triple block copolypeptides were prepared in mushroom derived tyrosinase treated or bacterial co-expression systems. First, 10 mM phosphate buffer (pH 5) containing 10 mM and 100 mM NaIO ₄ was dissolved in 10, 20 and 30% by weight of EBPPI [G1A4F1] 6-MFP [Mgfp5] 1 double block copolypeptides. The adherent surface was rinsed with acetone, ethanol and water. Each block copolypeptide solution was placed directly on the attachment and mixed with ₁₀ mM NaIO ₄ oxidant. The deposits were covered with other deposits to form overlapping regions and cured at 4 ° C. for 1 hour. Surface adhesion was hardened by immersion in DW at 25 ° C., and then EBPPI [G ₁ A ₃ F ₂ ] ₁₂ -EBPP [A ₁ G ₄ I ₁ ] ₆ -EBPPI [G ₁ A ₃ F ₂ ] ₁₂ Compared to the polypeptide.

As a result, as shown in FIG. 10 (B), 30 wt% double block copolypeptide treated with 100 mM NaIO ₄ exhibited chemical gelation due to crosslinking between quinone-mediated molecules. However, it showed lower surface adhesion than double block copolypeptides treated with ₁₀ mM NaIO ₄ (FIG. 10 (C)). Therefore, it means that DOPA molecule of the dual block co polypeptides treated with 100mM NaIO ₄ is that the more the oxidation proceeds to quinone than those treated with 10mM NaIO _4.

In addition, EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ -EBPPI [G ₁ A ₄ F ₁ ] ₆ triple block copolypeptide (10% by weight) was treated with 10 mM NaIO ₄ , the control EBPPI [G ₁ A ₃ F ₂ ] ₁₂ -EBPP [G ₁ A ₄ F ₁ ] ₆ -EBPPI [G ₁ A ₃ F ₂ ] _{12 It} was confirmed that the surface adhesion is stronger than the triple block copolypeptide. This is because the MFP intermediate block has surface adhesion (FIG. 11).

_{Also, EBPPI [G 1 A 4 F} 1] 6 -MFP [Mgfp5] 1 -EBPPI [G 1 A 4 F 1] 6 surface adhesion strength of the triple-block copolyester peptides _{EBPPI [G 1 A 4 F 1} ] 6 - It was found to be superior to MFP [Mgfp5] ₁ double block copolypeptides. This is due to the cohesion induced by the EBPPI blocks physically aggregated via crosslinking.

Example 9 Characterization of Block Copolypeptides Expressed by Bacterial Co-Expression and Confirmation of Hydrogel Adhesion

Hydroxylated block copolypeptides were prepared in various weight percents in a bacterial co-expression system. First, 10% by weight of EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptide was dissolved in 10 mM phosphate buffer (pH 5). Each block copolypeptide solution was mixed with 10 mM-100 mM NaIO ₄ oxidant prior to placing the attachment. Each block copolypeptide was placed on an attachment and then covered with another attachment to form an overlapped region and cured for 1 hour at 4 ° C.

As a result, as shown in FIG. 12 (A), surface adhesion of 10% by weight of the hydroxylated EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptide in a bacterial co-expression system It was confirmed that the attachments strongly bound by sex. In addition, this deposit also showed strong adhesion in the aquatic environment (FIG. 12 (B)).

When comparing the adhesion performance of hydroxylated double block copolypeptides in a bacterial co-expression system with a bacterial-derived tyrosinase treated double block copolypeptide at 10% by weight, It was found that the surface adhesion of the polypeptide is stronger.

Thus, the hydroxylation rate of tyrosine residues was increased in the case of hydroxylated double or triple block copolypeptides in a co-expression system, so that the same block copolypeptides would be hydroxylated in a bacterial co-expression system. Not only does it show strong surface adhesion, but it is also useful for industrial scale-up.

Example 10: Characterization and Surface Adhesion of Core-Shell Structures of Double and Triple Block Copolypeptides

The properties of the core-shell structure of EBP-MFP block copolypeptides with or without hydroxyl groups were analyzed by dynamic light scattering (DLS) instruments (Malvern instruments, Worcestershire, UK). The hydrodynamic radius (R _h ) of the 12.5 μM block copolypeptide in 10 mM phosphate buffer (pH5) was measured for 11 min after equilibration at 10 ^o C and 45 ^o C for 1 min.

As a result, as shown in (c) of FIG. 13 (A), EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptide with mushroom-derived tyrosinase-catalyzed hydroxylation. of 10 ^o for a hydrodynamic radius from the measured C, it was found to have a hydrodynamic radius of 25nm ~ 30nm at 10 ^o C. This confirms that due to the cohesion of the MFP molecules, the hydrophobic interaction of the MFP molecules is greatly increased, thereby forming a structure composed of the core of the MFP (Figs. 14 (A), 14 (B) and 14 (C)). ). Also shown in (d), hydrodynamics of EBPPI [G ₁ A ₄ F ₁ ] ₆ -MFP [Mgfp5] ₁ double block copolypeptides with mushroom-derived tyrosinase-catalyzed hydroxylation at 45 ^° C. As a result of measuring the radius, it was confirmed that the hydrodynamic radius of 50nm ~ 60nm. This confirms that the EBP block, which was soluble at or below the transition temperature, forms a core-shell structure by forming a hydrophobic core by phase transition above the transition temperature (Figs. 14 (G), 14 (H) and 14 (I). )).

Also, in order to confirm surface adhesion of the core-shell structure of the EBP-MFP block copolypeptide, an experiment was performed on the surface adhesion using hydrophobic fluorescent dyes.

In order to confirm the surface adhesion of the core-shell structure, a rhodamine 6G fluorescent dye with a final concentration of 0.5 w / v% was used. The fluorescent dyes were each (1) nothing in the PCR tube (# PCR-02-C, Axygen), (2) mixed with EBP double block copolypeptides, and (3) EBPPI hydroxylated via coexpression [G ₁ A ₄ F ₁ ] _6- MFP [Mgfp5] ₁ diblock copolypeptide mixed at 10 ^° C., and (4) EBPPI [G ₁ A ₄ F ₁ ] hydroxylated via coexpression _A mixture of _6- MFP [Mgfp5] ₁ double block copolypeptide at 40 ^° C. was used. Each block copolypeptide is 25 uM dissolved in 10 mM phosphate buffer (pH5). In addition, in the case of (2), (3), (4) to raise the pH to 8 using sodium hydroxide, and then stabilize for 3 hours at 25 ^o C, 50 ^o C, 10 ^o C, 40 ^o C, respectively. And all the tubes were washed with distilled water and ethanol.

As a result, as shown in Fig. 13 (B), only the tube of (4) was confirmed that the band of the color of the fluorescent dye. When the block copolypeptide forms a hydrophobic core, the hydrophobic fluorescent dye binds via hydrophobic interaction with the polypeptide of the core. Accordingly, in the case of (2), the EBP block forming the hydrophobic core and the fluorescent dye are present in combination, but since there is no surface adhesion, all of the core-shell structured EBP double block copolypeptides containing the fluorescent dye are washed. It is impossible to check the fluorescent dye by washing off. In the case of (3), since the hydrophobic core is composed of the MFP block, the fluorescent dye interacts with the MFP block, but the surface adhesion is greatly reduced by the MFP block present in the core. Could not be observed. Case (1) was performed to investigate the nonspecific reaction between the fluorescent dye and the tube during stabilization in the tube. As a result, the fluorescent dye could not be observed after washing. In the case of (4), the hydrophobic core of the EBP block interacts with the fluorescent dye at the transition temperature and the surface is adhered by the hydroxylated MFP block through co-expression in the shell. It was confirmed that the fluorescent dye had a color later.

As described above in detail specific parts of the present invention, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (21)

1. Elastin-based peptides (EBPs); And

   Multiblock copolypeptide consisting of mussel murine protein (MFP).
2. The method of claim 1, wherein the multiblock copolypeptide comprises (EBP) n (MFP) n; (EBP) n (MFP) n (EBP) n; And (MFP) n (EBP) n (MFP) n, wherein the array is any one of n, wherein n is an integer of 1 or more, and the number of repetitions of EBP or MFP is repeated.
3. The elastin-based peptide (EBP) of claim 1, wherein the elastin-based peptide (EBP) comprises: [VPGXG VPGXG VPGXG VPGXG VPGXG VPGXG] block; [VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG] block; And [IPAXG IPAXG IPAXG IPAXG IPAXG IPAXG IPAXG] A multi-block copolypeptide, characterized in that the amino acid sequence represented by any one selected from the group consisting of blocks, wherein X is an amino acid except proline.
4. The X of the [VPGXG VPGXG VPGXG VPGXG VPGXG VPGXG] block,

   A (Ala), G (Gly), I (Ile) consists of a ratio of 1: 4: 1;

   K (Lys), G (Gly), I (Ile) are in a ratio of 1: 4: 1;

   Or D (Asp), G (Gly), I (Ile) in a ratio of 1: 4: 1;

   E (Glu), G (Gly), I (Ile) consists of a ratio of 1: 4: 1;

   G (Gly), A (Ala), F (Phe) consists of 1: 3: 2 ratio;

   K (Lys), A (Ala), F (Phe) consists of a ratio of 1: 3: 2;

   D (Asp), A (Ala), F (Phe) consists of a ratio of 1: 3: 2;

   K (Lys), F (Phe) consist of 3: 3 ratio;

   D (Asp), F (Phe) consist of 3: 3 ratio;

   H (His), A (Ala), I (Ile) are in a ratio of 3: 2: 1;

   H (His), G (Gly) is in the ratio of 5: 1; or

   G (Gly), C (Cys), F (Phe) is a multi-block copolypeptide, characterized in that the ratio of 1: 3: 2.
5. 4. The X according to claim 3, wherein X in the [VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG] block is

   A (Ala), G (Gly), I (Ile) consists of a ratio of 1: 4: 1;

   K (Lys), G (Gly), I (Ile) are in a ratio of 1: 4: 1;

   Or D (Asp), G (Gly), I (Ile) in a ratio of 1: 4: 1;

   E (Glu), G (Gly), I (Ile) consists of a ratio of 1: 4: 1; or

   G (Gly), A (Ala), F (Phe) is a multi-block copolypeptide, characterized in that the ratio of 1: 3: 2.
6. 4. The X according to claim 3, wherein X in the [IPAXG IPAXG IPAXG IPAXG IPAXG IPAXG] block is

   G (Gly), A (Ala), F (Phe) is a multi-block copolypeptide, characterized in that the ratio of 1: 4: 1 or 1: 3: 2.
7. According to claim 1, wherein the mussel musa protein (MFP) is a mussel californianus foot protein 5 (Mcfp5) or the mussel galloprovincialis foot protein 5 (Mgfp5) characterized in that Multiblock Copolypeptides.
8. The multiblock copolypeptide of claim 1, wherein the multi-block copolypeptide is represented by an amino acid sequence represented by SEQ ID NO: 50 to 70.
9. The gene encoding the multiblock copolypeptide of claim 1.
10. Recombinant vector comprising the gene of claim 9.
11. A recombinant microorganism into which the gene of claim 9 or the recombinant vector of claim 10 is introduced.
12. The recombinant microorganism of claim 11, wherein an expression vector comprising a gene encoding tyrosinase or a gene encoding tyrosinase is further introduced and co-expressed.
13. Method for preparing a multiblock copolypeptide comprising the following steps:

    (a) culturing the recombinant microorganism of claim 11 to produce a multi-block copolypeptide; And

    (b) obtaining the resulting multiblock copolypeptides.
14. The method according to claim 13, wherein the recombinant microorganism of step (a) is further introduced with an expression vector comprising a gene encoding a tyrosinase (tyrosinase) or a gene encoding a tyrosinase and the multi-block copolypeptide A method for producing tyrosinase, which is co-expressed.
15. The method of claim 14, wherein the tyrosine residue of the multiblock copolypeptide is modified by the tyrosinase to a dopa (3,4-dihydroxyphenylalanine) residue.
16. The self-assembled nanostructure of the core-shell structure according to any one of claims 1 to 8, wherein the EBP block forms a core structure and the MFP block forms a shell structure by temperature stimulation.
17. A drug delivery composition comprising the self-assembled nanostructure of claim 16.
18. The hydrogel of claim 1, wherein the multiblock copolypeptides of claim 1 are prepared by forming crosslinks between block polypeptides by temperature stimulation.
19. 19. The hydrogel of claim 18, wherein the hydrogel is formed by oxidative or non-covalent interaction of dopa (3,4-dihydroxyphenylalanine) residues contained in mussel murine protein (MFP).
20. A bioadhesive composition comprising the hydrogel of claim 18.
21. Surgical suture comprising a hydrogel of claim 18.

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