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KR20160018234A - Composition comprising recombinant protein derived from sea anemone for preparing hydrogel and method for preparing hydrogel using the same - Google Patents

Composition comprising recombinant protein derived from sea anemone for preparing hydrogel and method for preparing hydrogel using the same Download PDF

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KR20160018234A
KR20160018234A KR1020140102544A KR20140102544A KR20160018234A KR 20160018234 A KR20160018234 A KR 20160018234A KR 1020140102544 A KR1020140102544 A KR 1020140102544A KR 20140102544 A KR20140102544 A KR 20140102544A KR 20160018234 A KR20160018234 A KR 20160018234A
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recombinant protein
gly
derived
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protein
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차형준
양윤정
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포항공과대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
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    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
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    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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    • C08L67/00—Compositions of polyesters obtained by reactions forming a carboxylic ester link in the main chain; Compositions of derivatives of such polymers
    • C08L67/04—Polyesters derived from hydroxycarboxylic acids, e.g. lactones
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    • C08L71/00—Compositions of polyethers obtained by reactions forming an ether link in the main chain; Compositions of derivatives of such polymers
    • C08L71/02—Polyalkylene oxides
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    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L89/00—Compositions of proteins; Compositions of derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand

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Abstract

The present invention relates to a method of preparing recombinant proteins derived from sea anemone having similar sequential, structural characteristics to collagen or silk, to a composition for preparing protein-based hydrogel comprising the same, to a gel comprising crosslinked product of the recombinant proteins derived from sea anemone, and to a preparation method of the gel. According to the present invention, the recombinant proteins derived from sea anemone can be mass-produced and separated by a genetic engineering method while having excellent mechanical properties, and can retain water while maintaining a three-dimensional structure. Therefore, the recombinant proteins in the present invention can be applied for field of medical materials including artificial biological membranes such as cornea and eardrum, and have potential applicability as novel protein-based materials.

Description

TECHNICAL FIELD The present invention relates to a composition for preparing a hydrogel comprising an anemia-derived recombinant protein and a method for producing the hydrogel including the recombinant protein derived from the sea anemone.

The present invention relates to a composition for preparing a hydrogel comprising an anthrax-derived recombinant protein, a method for producing the hydrogel using the same, and a hydrogel comprising the same.

A hydrogel means a hydrophilic, expansible polymer network, usually having a water content of 30% or more, and a water absorbent hydrogel having a water content of 90% or more. Since hydrogels can capture water and have a stable three-dimensional structure, they have a high advantage as a basic support for cell culture, and thus biotechnological application researches have been actively conducted.

As the poly-2-hydroxyethyl methacrylate (PHEMA) was known in the 1960's, studies on hydrogelation based on chemosynthetic polymers were started. After 1980, hydrogel using calcium alginate was developed, The interest in polymeric materials has increased. Thus, hydrogels can be divided into natural hydrogels and synthetic hydrogels depending on the basic materials used in production, Natural hydrogels include protein-based hydrogels including collagen, elastin, fibroin, and silk, polysaccharide-based hydrogels including HA, alginate, chitosan, dextran, and DNA-based hydrogels using plasmids .

In the case of synthetic hydrogels, biodegradable PEG, PPF-PEG, and PHEMA-PCL are representative examples, and PHEMA, PHPMA, PNIPAm, PEGDA, and PVA, which are not biodegradable, are also widely used. Or blending them to produce various hybrid hydrogels to produce hydrogels with better properties.

Or bioreactor properties, it is possible to prepare hydrogels capable of reacting with specific biomolecules such as cells, enzymes, growth factors and the like. Reactive hydrogels that specifically react to physical stimuli (temperature, light, pressure, electrical) or chemical stimuli (ions, pH, molecular recognition) can also be used as sensors, diagnostics, microchannels, . Particularly in the production of hydrogels applicable to bioengineering research, reactivity with cells, adhesion and proliferation of cells are important factors. Therefore, by introducing functional peptides of cell adhesion ability, the reaction with cells can be promoted. For example, fibronectin-derived (RGD, EILDV, LIGRKK, SPPRRARV, WQPPRARI, KQAGDV, etc.), vitronectin-derived (GKKQRFRHRNRKG), bone sialoprotein-derived (FHRRIKA), laminin derived (IKVAV, YIGSR, PDGSR, LRGDN, LRE, IKLLI, (DGEA, GFOGER, GDR, GRD), etc., and prior studies for introducing such functional moieties into polymers based on chemical synthesis are already well known.

Protein-based polymers have a higher biodegradability and better reactivity with cells than chemical polymers, so that the importance of recombinant proteins or natural extract protein-based hydrogels is increasing. However, compared to polymer-based hydrogels, It has a disadvantage that its physical properties are weak.

Mechanical properties are important for the hydrogel to maintain its shape during the regeneration of living tissue in a three-dimensional space. The mechanical properties of the hydrogel may depend on various factors such as the concentration of the protein, the crosslinking method, the type of crosslinking agent, the concentration of the crosslinking agent, and the post-treatment method. In addition, the mechanical properties induced by the choice of protein material for hydrogels are also different. For example, the mechanical properties of silk or collagen-based hydrogels, which are structural proteins, are relatively high. Nevertheless, the various searches of gel materials to increase mechanical properties were relatively inadequate.

Collagen, a typical example of structural proteins, is the main component of the connective tissue that constitutes our body and is the basic skeleton of the extracellular matrix. Although collagen can be obtained through extraction in nature, there is a problem that it is required to pay a high price due to the problem of heterologous immunity derived from other animals and the difficulty of protein extraction and separation. As an alternative, attempts have been made to produce recombinant collagen, but it is difficult to artificially simulate the unique triple helical structure of collagen and the modified hydroxyproline.

Another structural protein, silk protein, can be obtained in a large amount in silkworm, but it has lower physical strength than spider silk. In the case of spider silk, it is impossible to mass-feed due to the nature of spiders eating each other, and there is a limit to obtaining large quantities naturally. The repetitive sequences of silkworms and spiders are large in size and are not easy to produce as recombinant proteins under the E. coli system. Therefore, many studies have attempted to express a part of the repeating sequence that affects the physical properties, or to improve the physical properties through the crosslinking method after their expression.

In the 2000s, as silk proteins were known to have different physical properties depending on species and species, studies on silk derived from silkworms and spiders from various species began to be reported. In the case of spiders, silk research limited to dragline silk was extended to flagelliform, cylindriform. In addition, it has been found that silk or silk-like proteins are present in marine organisms such as spiders, bees, ants, dragonflies, and grasshoppers, as well as silkworms and spiders, and mussels, pearl shells, Although various proteins of a new structure and sequence are known, insects and marine organisms can not be cultivated, and the amount of silk protein obtained is insufficient, so that it is not possible to utilize it as a natural extract. As an alternative, recombinant protein production technology is one of the alternatives to discover and apply novel proteins that exist in nature but are difficult to express. There have already been successful cases of production of enzymes, antibodies and useful proteins for medical and industrial purposes. Using this technique, the chain length of high molecular weight proteins having a size of 100 kDa or more can be freely controlled in living organisms such as Escherichia coli, yeast or animal cells. Expression and production of a repeat sequence protein based on recombinant protein production technology can not only utilize naturally occurring useful genetic sequences but also reveal various structures and characteristics of unknown proteins. Above all, it can be a method to elicit more improved mechanical properties in the production of protein-based hydrogels which showed limitations in physical properties.

Research on protein-based materials has already been conducted in advanced countries such as the United States, Australia, and Japan. The development of existing protein materials has been promoted through the production of silk-elastin fusion proteins, as well as the discovery of new proteins such as antisole and resilin. The discovery and application of new genetic sequences is not only based on structural, mechanical, and physicochemical properties that are not known to the basics, but it can also broaden the application area, have. Above all, little information and applications on living organisms in the oceans reveal the importance and high potential of excavating new materials derived from marine-derived organisms.

In the present invention, novel sequences based on anthocyanidase-derived silk-like proteins and collagen-like proteins have been searched. As a result, it has been found that repetitive sequence recombinant proteins are expressed and purified, the preparation of the material is achieved through gelation of the proteins, Thereby completing the present invention.

Accordingly, an object of the present invention is to provide a composition for preparing a gel comprising at least one protein selected from the group consisting of silk-like silk-like proteins and collagen-like proteins.

It is still another object of the present invention to provide a gel production method comprising crosslinking the composition.

A further object of the present invention is to provide a gel comprising a crosslinked product of said protein.

One embodiment of the present invention relates to a composition for preparing a gel comprising an anthrax-derived silk-like recombinant protein and / or a collagen-like recombinant protein, a method for producing a gel using the anthrax-derived silk-like recombinant protein and / will be.

In the present invention, hydrogels were prepared based on the silk and collagen-like proteins mainly present in the tentacles and skin of an anemone, and the possibilities as biomaterials for biotechnology were confirmed. The anthrax-derived silk-like protein has been identified as a flagelliform, a type of spider silk protein, and an anthrax-derived collagen-like protein, has a sequence and structural homology with collagen. The anthrax-derived silk-like protein contains about 5% of tyrosine and the anthrax-derived collagen-like protein has a high tyrosine content of about 10%. Therefore, it is possible to induce di-tyrosine residues through photo-crosslinking of proteins based on tyrosine. Such induction of covalent bonds enables gelation of protein-based hydrogels and induces higher mechanical properties It can be a way to do that.

Specifically, the present invention provides a composition for preparing a gel comprising an anemone-derived recombinant protein, for example, at least one protein selected from the group consisting of an anthrax-derived silk-like recombinant protein and a collagen-like recombinant protein.

The collagen-like recombinant protein may be a recombinant protein having at least one repetitive unit selected from the group consisting of repeating units having the amino acid sequence of SEQ ID NO: 1, for example, 5 to 120 repeats, preferably 5 to 120 repeats The present invention relates to a composition for preparing a gel comprising a polypeptide, wherein the polypeptide comprises a protein linked repeatedly 1 to 20 times.

In addition, the silk-like recombinant protein may be produced by repeating a total of 1 to 200 repeats, preferably 5 to 120 repeats, of at least one repeating unit selected from the group consisting of repeating units having the amino acid sequence of SEQ ID NO: Wherein the polypeptide comprises a protein linked repeatedly from 1 to 20 times.

The repeating unit of the collagen-like recombinant protein may be selected from the group consisting of the amino acid sequences of SEQ ID NOS: 2 to 33, preferably the repeating unit group consisting of the amino acid sequences of SEQ ID NOS: 2, 8, 10, 18, Lt; / RTI >

The repeating unit of the silk-like recombinant protein may be selected from the group consisting of the amino acid sequences of SEQ ID NOS: 37 to 68, preferably the repeating unit selected from the group consisting of the amino acid sequences of SEQ ID NOS: 37, 38, Lt; / RTI >

[SEQ ID NO: 1]

GL-Xaa 1 -V-Xaa 2 -Y-Xaa 3 -P-Xaa 4 -Xaa 5

In this sequence, Xaa 1 = P or Q, Xaa 2 = L or F, Xaa 3 = P or T, Xaa 4 = S or T, or Xaa 5 = N or I.

[SEQ ID NO: 36]

Xaa 1 -Xaa 2 -Xaa 3 -NTG-Xaa 4 -P-Xaa 5 -Q

In the above sequence, Xaa 1 = G or D, Xaa 2 = P or S, Xaa 3 = G or S, Xaa 4 = Y or C, or Xaa 5 = G or W.

The anthrax-derived collagen-like recombinant protein may be a polypeptide having an amino acid sequence of SEQ ID NO: 1 linked at least once, preferably a polypeptide linked between 1 and 200 times, more preferably between 1 and 120 times. Also, the polypeptide may be a polypeptide having another amino acid sequence and being linked between 1 and 200 times or between 1 and 120 times.

For example, the collagen-like recombinant protein includes an amino acid sequence of SEQ ID NO: 34 or 35, and has an 80% or more sequence homology with the amino acid sequence of SEQ ID NO: 34 or 35 to provide an anthrax-derived collagen-like recombinant protein do. Preferably an anthaxe-derived collagen-like recombinant protein characterized by having 90% or more sequence homology with the amino acid sequence set forth in SEQ ID NO: 34 or 35.

The anthrax-derived silk-like recombinant protein may be a polypeptide linked to the amino acid sequence of SEQ ID NO: 36 at least once, preferably a polypeptide linked between 1 and 200 times, more preferably between 1 and 120 times. Also, the polypeptide may be a polypeptide having another amino acid sequence and being linked between 1 and 200 times or between 1 and 120 times.

For example, the silk-like recombinant protein comprises the amino acid sequence of SEQ ID NO: 69 or 70, and has an 80% or more sequence homology with the amino acid sequence of SEQ ID NO: 69 or 70. The silk- Protein. Preferably an anthrax-derived silk-like recombinant protein having a sequence homology of 90% or more with the amino acid sequence set forth in SEQ ID NO: 69 or 70. [

One embodiment of the present invention is to provide a gel production method comprising crosslinking the anthrax-derived silk-like recombinant protein and / or the collagen-like recombinant protein.

A further embodiment of the present invention is to provide a gel comprising a cross-linked silk-like recombinant protein and / or a collagen-like recombinant protein derived from said sea anemone.

Protein formulation is achieved through gelation of silk-like recombinant proteins and / or collagen-like recombinant proteins derived from anthaxia, and based on their mechanical properties, biodegradable proteins such as cell-supporting fixtures, microfilters, enzymes, drug carriers, Based functional medical material.

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for preparing a gel comprising an anthrax-derived silk-like recombinant protein and / or a collagen-like recombinant protein, a process for producing a gel comprising crosslinking the recombinant protein, and a gel comprising a crosslinked product of the recombinant protein .

The present invention provides a composition for preparing a gel comprising at least one protein selected from the group consisting of an anthrax-derived silk-like recombinant protein and a collagen-like recombinant protein. Specifically, the composition for preparing a gel according to the present invention may comprise a single component of each of anthocyanin-derived silk-like recombinant protein or collagen-like recombinant protein or a mixture thereof.

The anthrax-derived collagen-like recombinant protein of the present invention comprises at least one member selected from the group consisting of repeating units consisting of amino acid sequences having the general formula of SEQ ID NO: 1, and the total number of repeating units is 1 to 200, preferably 5 To 150 times, for example, 10 to 120 times, and the polypeptide is a protein that is repeatedly attached by repeating 1 to 20 times.

The repeating unit having the general formula of SEQ ID NO: 1 may be selected from the group consisting of repeating units having the amino acid sequence of SEQ ID NOS: 2 to 33, preferably at least one of SEQ ID NOS: 2, 8, 10, 18, 19 < / RTI > amino acid sequence.

Considering that the molecular weight of the natural-derived collagen fiber is about 2 to 300 kDa, the increase in the number of repeats of the repeated sequence of the recombinant protein can be a method of simulating natural collagen. Above all, a material having a high molecular weight can increase the mechanical properties, viscosity, toughness, durability, radioactivity, rubber elasticity and the like.

The anthrax-derived collagen-like recombinant protein according to the present invention is a hypothetical protein, and information of NCBI (NCBI accession number XP_001623116.1) is used. In the case of the anthrax-derived collagen protein in nature, It has 198 amino acids.

The anthrax- derived collagen-like recombinant protein of the present invention can be used for Nematostella the recombination of the codon sequences of some of the mRNA sequences of an anemia-derived collagen protein encoding the amino acid sequence derived from vectensis and optimizing the molecular weight to prepare an expression vector containing the optimized 190 amino acid repeat sequence protein gene, A vector is inserted into a host cell to produce a transformant, which is then produced from the transformant.

Triple helical structure of collagen present in the sea anemone-derived collagen, similar to the recombinant protein of the present invention are bacteria (Syntrophobotulus glycolicus), mussels (Mytilus californianus ) have 48% and 32% identity, respectively.

An anthrax-derived collagen-like recombinant protein according to the present invention is characterized by having a His tag, a GST tag, and a maltose binding protein tag at the C-terminus, N-terminus, or both ends of the protein , And may include a restriction enzyme recognition site or a part thereof at the C-terminus, N-terminus, or both ends of the recombinant protein during the production of the recombinant protein. A specific example of the recombinant protein according to an embodiment of the present invention is that when a recombinant protein is produced from the expression vector, 6 histidine is connected to the C-terminal of the protein (6 x His-tagged tag sequence: HHHHHH) Respectively.

One embodiment of the present invention may include a polypeptide in which one or more repeating units selected from the repeating unit group consisting of the amino acid sequence of SEQ ID NO: 1 is repeatedly repeated for a total of 1 to 200 repetitions. The total number of repeating units of the repeating unit may include a polypeptide consisting of 10 to 1200 amino acids repeatedly, preferably 1 to 120 times in total, and the polypeptide is a protein linked repeatedly 1 to 20 times. And may further include a polypeptide to which another amino acid sequence is further included at a portion where the repeating units are connected to each other. The amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of an anthrax-derived collagen-like recombinant protein and has the following general formula.

[Formula I]

GL-Xaa 1 -V-Xaa 2 -Y-Xaa 3 -P-Xaa 4 -Xaa 5 (SEQ ID NO: 1)

Xaa 1 is P or Q, Xaa 2 is L or F, Xaa 3 is P or T, Xaa 4 is S or T, and Xaa 5 is N or I. In the above formula I,

 Specific examples of the repeating unit having the amino acid sequence of SEQ ID NO: 1 are shown in SEQ ID NO: 2 to SEQ ID NO: 33 and are shown in Table 1 below.

SEQ ID NO: Amino acid sequence (N-terminal to C-terminal) SEQ ID NO: 2 GLPVLYPPSN SEQ ID NO: 3 GLPVLYPPSI SEQ ID NO: 4 GLPVLYPPTN SEQ ID NO: 5 GLPVLYPPTI SEQ ID NO: 6 GLPVLYTPSN SEQ ID NO: 7 GLPVLYTPSI SEQ ID NO: 8 GLPVLYTPTN SEQ ID NO: 9 GLPVLYTPTI SEQ ID NO: 10 GLPVFYPPSN SEQ ID NO: 11 GLPVFYPPSI SEQ ID NO: 12 GLPVFYPPTN SEQ ID NO: 13 GLPVFYPPTI SEQ ID NO: 14 GLPVFYTPSN SEQ ID NO: 15 GLPVFYTPSI SEQ ID NO: 16 GLPVFYTPTN SEQ ID NO: 17 GLPVFYTPTI SEQ ID NO: 18 GLQVLYPPSN SEQ ID NO: 19 GLQVLYPPSI SEQ ID NO: 20 GLQVLYPPTN SEQ ID NO: 21 GLQVLYPPTI SEQ ID NO: 22 GLQVLYTPSN SEQ ID NO: 23 GLQVLYTPSI SEQ ID NO: 24 GLQVLYTPTN SEQ ID NO: 25 GLQVLYTPTI SEQ ID NO: 26 GLQVFYPPSN SEQ ID NO: 27 GLQVFYPPSI SEQ ID NO: 28 GLQVFYPPTN SEQ ID NO: 29 GLQVFYPPTI SEQ ID NO: 30 GLQVFYTPSN SEQ ID NO: 31 GLQVFYTPSI SEQ ID NO: 32 GLQVFYTPTN SEQ ID NO: 33 GLQVFYTPTI

The repeating units constituting the anthrax-derived sea anemone-derived anthrax-like recombinant protein of the present invention are preferably SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 18 and SEQ ID NO: 19, May be repeated independently and the total number of repetitions may be 5 to 200 times, preferably 10 to 120 times.

When the anthrax-derived collagen-like recombinant protein contains a histidine tag, a slight molecular weight change occurs. As a result of checking the size of the protein under electrophoresis (SDS-PAGE), the presence of the protein was confirmed at a molecular weight slightly smaller than the original size of the target protein. These results suggest that protein migration during electrophoresis may be different due to the amino acid sequence of collagen - like recombinant proteins derived from anthracnose. In order to identify the exact size of the anthrax-derived collagen-like recombinant protein, MALDI-MS analysis was performed to confirm the molecular weight corresponding to the theoretical value. However, the solubility of protein in water or buffer was poor, Due to its nature, it inhibited the crystal formation of the matrix, which impeded the ion desorption by the laser and was not easy to analyze.

The anthrax-derived silk-like recombinant protein of the present invention comprises at least one member selected from the group consisting of repeating units consisting of the amino acid sequence having the general formula of SEQ ID NO: 36, wherein the total number of repeating units is 1 to 200 times, 5 to 150 times, for example, 10 to 120 times, and the polypeptide may be one in which the other amino acid sequence is included in between 1 and 200 times or between 1 and 120 times.

Considering that the molecular weight of the natural-derived silk fiber is about 2 to 500 kDa, the increase in the number of repeats of the repeated sequence of the recombinant protein can be a method of simulating natural-derived silk. Above all, since a substance having a high molecular weight exhibits properties of a polymer, higher mechanical properties, viscosity, toughness, durability, radioactivity, and rubber elasticity can be increased.

The repeating unit having the general formula of SEQ ID NO: 36 may be selected from the group consisting of repeating units having the amino acid sequence of SEQ ID NOs: 37 to 68, preferably at least one of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: And the amino acid sequence of SEQ ID NO: 47.

Anomalous silk proteins in nature have a repeating structure of five amino acids [GPGXX (X = A, V, S, Y)], known as flagelliform spider origins, As the protein (amino acid sequence of SEQ ID NO: 69), the information of NCBI Reference Sequence (XP_001621085.1) was used.

The anthrax- derived silk-like recombinant protein of the present invention can be used for Nematostella a transformant is prepared by preparing an expression vector by optimizing a 190 amino acid repeat sequence in which a repeating structure appears in an amino acid sequence derived from a vector, and then introducing the expression vector into a host cell to produce a transformant and then producing the transformant.

The anthocyanin silk protein obtained from the NCBI search was a silkworm ( Bombyx mori ), 49.5% identity and 60.2% homology to the silk protein from the spider ( Nephila clavipes- derived silk protein with 43.2% identity and 64% homology.

The anthrax- derived silk-like recombinant protein according to the present invention was prepared by rearranging codon sequences of mRNA sequences coding for the amino acid sequence of an imaginary repeat sequence protein derived from anemone ( Nematostella vectensis ) An expression vector containing the gene of the recombinant protein is prepared, the expression vector is inserted into a host cell to produce a transformant, and then the transformant is produced.

The anthrax-derived silk-like recombinant protein according to the present invention is characterized in that a His tag, a GST tag, and an MBP tag (maltose binding protein tag) are attached to the C-terminus, N-terminus, , And may include a restriction enzyme recognition site or a part thereof at the C-terminus, N-terminus, or both ends of the recombinant protein during the production of the recombinant protein. A specific example of the recombinant protein according to one embodiment of the present invention is that when a recombinant protein is produced from the expression vector, 6 histidine is connected to the C-terminal of the protein (6 x His-tagged tag sequence; HHHHHH) (MKAIFVLKDDDDK). ≪ / RTI > This is represented by SEQ ID NO: 70.

One embodiment of the present invention may include a polypeptide in which at least one repeating unit selected from the repeating unit group consisting of the amino acid sequence of SEQ ID NO: 36 is repeatedly linked to the total number of repeats 1 to 200 times. The total number of repeating units of the repeating unit may include a polypeptide consisting of 10 to 1200 amino acids repeatedly, preferably 1 to 120 times in total, and the polypeptide is a protein linked repeatedly 1 to 20 times. And may further include a polypeptide to which another amino acid sequence is further included at a portion where the repeating units are connected to each other. The amino acid sequence of SEQ ID NO: 36 is the amino acid sequence of an anthrax-derived silk-like recombinant protein and has the following general formula.

 [Formula 2]

Xaa 1 -Xaa 2 -Xaa 3 -NTG-Xaa 4 -P-Xaa 5 -Q (SEQ ID NO: 36)

In the above sequence, Xaa 1 = G or D, Xaa 2 = P or S, Xaa 3 = G or S, Xaa 4 = Y or C, or Xaa 5 = G or W.

Specific examples of the repeating unit having the amino acid sequence of SEQ ID NO: 36 are shown in SEQ ID NOS: 37 to 68 and are shown in Table 2 below.

SEQ ID NO: Amino acid sequence (N-terminal to C-terminal) SEQ ID NO: 37 GPGNTGYPGQ SEQ ID NO: 38 DPGNTGYPGQ SEQ ID NO: 39 GSGNTGYPGQ SEQ ID NO: 40 DSGNTGYPGQ SEQ ID NO: 41 GPSNTGYPGQ SEQ ID NO: 42 DPSNTGYPGQ SEQ ID NO: 43 GSSNTGYPGQ SEQ ID NO: 44 DSSNTGYPGQ SEQ ID NO: 45 GPGNTGCPGQ SEQ ID NO: 46 DPGNTGCPGQ SEQ ID NO: 47 GSGNTGCPGQ SEQ ID NO: 48 DSGNTGCPGQ SEQ ID NO: 49 GPSNTGCPGQ SEQ ID NO: 50 DPSNTGCPGQ SEQ ID NO: 51 GSSNTGCPGQ SEQ ID NO: 52 DSSNTGCPGQ SEQ ID NO: 53 GPGNTGYPWQ SEQ ID NO: 54 DPGNTGYPWQ SEQ ID NO: 55 GSGNTGYPWQ SEQ ID NO: 56 DSGNTGYPWQ SEQ ID NO: 57 GPSNTGYPWQ SEQ ID NO: 58 DPSNTGYPWQ SEQ ID NO: 59 GSSNTGYPWQ SEQ ID NO: 60 DSSNTGYPWQ SEQ ID NO: 61 GPGNTGCPWQ SEQ ID NO: 62 DPGNTGCPWQ SEQ ID NO: 63 GSGNTGCPWQ SEQ ID NO: 64 DSGNTGCPWQ SEQ ID NO: 65 GPSNTGCPWQ SEQ ID NO: 66 DPSNTGCPWQ SEQ ID NO: 67 GSSNTGCPWQ SEQ ID NO: 68 DSSNTGCPWQ

The repeating unit constituting the anthrax-derived silk-like recombinant protein of the present invention is preferably SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 45 and SEQ ID NO: 47, wherein the above four repeating units are independently repeated And the total number of repetitions is 5 to 200 times, preferably 10 to 120 times.

When an anthropogenic silk-like recombinant protein contains a histidine tag, the protein behaves differently from its original size under electrophoresis (SDS-PAGE). This is because the amino acid sequences of silk-like recombinant proteins derived from anthracnose are different from each other in electrophoresis, or the protein appears as a dimer. This was confirmed by electrophoresis of proteins obtained by HPLC analysis.

An anthrax-derived collagen-like recombinant protein and a silk-like recombinant protein according to an embodiment of the present invention can be mass-produced by a genetic engineering method in a transformant by inserting an external gene into a commercially produced vector.

A method of inserting a gene encoding a target protein into an expression vector and inserting an expression vector into a host cell to produce a transformant for producing a recombinant protein can be carried out using a known established method.

Host cells used in the present invention include, but are not limited to, E. coli, yeast, animal cells, plant cells or insect cells. As the E. coli, for example, BL21 (DE3) may be used, but BLR (DE3) or BLR (DE3) pLysS may also be included as a selection strain, but the present invention is not limited thereto.

Hereinafter, the production of a transformant using E. coli as a host cell and the production method of the recombinant protein derived from anchovy are described in detail.

First, codon sequence rearrangement is performed to avoid repetition of the main codon in the mRNA sequence of an anemia-derived protein obtained from NCBI. The DNA sequence of an anemia-derived recombinant protein can be replaced with the most optimized codon in the host for expression and can be optimized to avoid codon duplication.

 The optimized DNA sequence of an anemia-derived recombinant protein was chemically synthesized and added to the Nde1 and Xho1 restriction sites at both ends for insertion into the expression vector. The designed recombinant proteins derived from an anemone are prepared by cloning into an expression vector pET23b (+) containing the expression promoter T7 (pT7).

An expression vector containing an anthrax-derived recombinant protein has a histidine tag [6 x his tag sequence; HHHHHH], and in the case of the anthrax-derived silk-like recombinant protein, an expression-enhancing peptide was added at the N-terminus. The expression vector is selected depending on the type of host cell and culture characteristics for protein production, and can be newly produced. Anemone-derived repeat sequence recombinant proteins produced according to the present invention preferably have the amino acid sequence of SEQ ID NO: 35 or 70.

The term "expression-enhancing peptide" refers to an amino acid sequence that enhances the level of expression of a recombinant protein by enhancing translation initiation efficiency, and is a translation initiation peptide comprising eight amino acids derived from the tryptophan operon of Escherichia coli .

The term "vector" means means for expressing a gene of interest in a host cell. For example, viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenovirus vectors, retroviral vectors and adeno-associated viral vectors. The vector that can be used as the recombinant vector may be a plasmid (for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14 , pGEX series, pET series, pUC19, etc.), phage (e.g., lambda gt4? B, lambda-Charon, lambda Delta z1 and M13 or viruses such as SV40).

The recombinant vector can typically be constructed as a vector for cloning or as a vector for expression. The expression vector may be any conventional vector used in the art to express an exogenous protein in plants, animals or microorganisms. The recombinant vector may be constructed by a variety of methods known in the art.

The recombinant vector may be constructed with prokaryotic or eukaryotic cells as hosts. For example, when the vector to be used is an expression vector and the prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (for example, pL λ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiation of translation and a transcription / translation termination sequence. When a eukaryotic cell is used as a host, the origin of replication that functions in eukaryotic cells contained in the vector includes f1 replication origin, SV40 replication origin, pMB1 replication origin, adeno replication origin, AAV replication origin, and BBV replication origin But is not limited thereto. Also, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, The cytomegalovirus promoter and HSV tk Promoter) can be used, and generally has a polyadenylation sequence as a transcription termination sequence.

The method for producing the protein more specifically comprises the steps of: preparing an expression vector comprising a polynucleotide encoding an anthrax-derived recombinant protein; Introducing the expression vector into a host cell to produce a transformant; Producing an anthrax-derived recombinant protein from said transformant; And separating and purifying the produced recombinant protein.

In an embodiment of the present invention, a transformant producing a recombinant protein can be prepared by inserting an expression vector into which a target protein is inserted into a host cell, and the transformant is obtained by introducing the recombinant vector into an appropriate host cell have. The host cell may be any host cell known in the art as a cell capable of continuously cloning or expressing the expression vector stably.

The host cells used in the present invention may include E. coli, yeast, animal cells, plant cells, insect cells, and the like. Examples of the prokaryotic cells include E. coli JM109, E. coli BL21, E. coli . Bacillus sp. strains such as E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, and Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcesens and There are various enterococci and strains such as various Pseudomonas species. When transforming eukaryotic cells, yeast ( Saccharomyce cerevisiae , insect cells, plant cells and animal cells such as Sp2 / 0, CHO ( Chinese hamster ovary ) K1, CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN and MDCK cell lines. The delivery (introduction) of the polynucleotide or the recombinant vector containing the polynucleotide into a host cell can be carried out by a method well known in the art. For example, when the host cell is a prokaryotic cell, the CaCl 2 method or the electroporation method can be used. When the host cell is a eukaryotic cell, the microinjection method, the calcium phosphate precipitation method, the electroporation method, Liposome-mediated transfection, and gene bombardment, but the present invention is not limited thereto.

The method of selecting the transformed host cells can be easily carried out according to a method widely known in the art by using a phenotype expressed by a selection marker. For example, when the selection mark is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

In one embodiment of the present invention, the production of a transformant using E. coli as a host cell and the method for producing the recombinant protein derived from anchovy are described in detail. As the E. coli, BLR (DE3), BL21 (DE3), or BLR (DE3) pLysS can be used, but the present invention is not limited thereto. The prepared expression vector is inserted into a host cell by thermal shock method to produce a transformant that produces an anthrax-derived recombinant protein. The transformant is inoculated on LB medium supplemented with ampicillin and cultured under shaking.

D-thiogalactopyranoside (IPTG), which is a protein expression inducing substance, when the absorbance (OD 600 ) of the culture broth is 0.4 to 1.0, preferably 0.8 to 1.0 at 600 nm in order to induce the expression of recombinant proteins derived from an anthracnose. To 0.1 to 10 mM, and further cultured. Cells are recovered from the culture.

The recovered cells are suspended in a lysis solution and disrupted using an ultrasonic disintegrator or a high-pressure disintegrator. A portion of the shredded cells are divided into whole cell lysate, soluble fraction and insoluble fraction, and the remaining whole cell lysate is centrifuged. Only the insoluble fraction is recovered, and the recovered fraction is separated and purified from an anemia-derived recombinant protein expressed using a Histag column. Specifically, recombinant affinity chromatography derived from an anemone produced in a host cell can be obtained by eluting the protein bound to the column. Preferably, the anomalous repeat sequence recombinant protein is disrupted with a cell disruption solution, the aqueous fraction of the disrupted cells is injected into a nickel NTA (nitrilotriacetic acid) column, and the protein not bound to the column is washed with the wash solution. The protein is separated from the column with elution buffer and purified.

In addition, anthocyanidase-derived collagen-like recombinant proteins can be more easily purified through acid extraction. Preferably, the insoluble fraction of the crushed cells containing the anthrax-derived collagen-like recombinant protein is separated by centrifugation, and the separated insoluble fraction is dissolved in an acid solution (for example, phosphoric acid or formic acid) Similar recombinant proteins can be extracted.

In addition, the anthrax-derived silk-like recombinant protein can be purified through thermal shock and acid extraction. Preferably, the aqueous fraction of the shredded cells containing the anthrax-derived silk-like recombinant protein is subjected to thermal shock at 40 to 100 DEG C, the resulting aqueous fraction is separated by centrifugation, the separated aqueous fraction is dissolved in an acid solution (For example, phosphoric acid or formic acid), and then removing the resulting insoluble fraction.

The purified recombinant proteins derived from an anemone, which is purified through affinity chromatography or thermal shock and acid extraction, are subjected to dialysis using water to remove components other than water and proteins remaining in the protein aqueous solution, and finally, A purified recombinant protein is obtained in powder form.

An anthrax-derived recombinant protein according to the present invention can be crosslinked to form a gel or a membrane, and can function as a gel-forming protein. Anemone-derived recombinant proteins can be made of gel-like three-dimensional materials, which requires protein cross-linking.

The protein gel can be prepared using a crosslinking method. Specifically, the protein gel derived from an anemone purified in the powder form is dissolved in at least one solvent selected from the group consisting of an acidic solvent and an organic solvent, Gelation of proteins can be induced by using a cross-linking method using pH, a protein cross-linking method using glutaraldehyde, or a photo-reactive cross-linking method, or the like.

An anthrax-derived recombinant protein can be gelled by mixing with a polymer compound. Examples of the polymeric compound include poly-ethylene oxide (PEO), polydioxanone (PDO), polycaprolactone (PCL), poly-lactic-co- glycolic acid (PLGA), poly-L-lactide acid (PLLA), chitosan But is not limited thereto.

In addition, an anthrax-derived recombinant protein can be gelled by mixing with albuminoids. Such albuminoids include, but are not limited to, keratin, gluten, lactalbumin, collagen, and recombinant proteins thereof.

The acidic solvent includes, but is not limited to, phosphoric acid, acetic acid, formic acid, hydrochloric acid, sulfuric acid, nitric acid, and citric acid. The organic solvent may include HFIP (hexafluoroisopropanol), HFP (hexafluoropropanol), HFA (hexafluoroacetone), TFA (trifluoroacetic acid), diisopropylethylamine, methylimidazolium chloride, It does not. In the present invention, formic acid is preferable. At this time, the concentration of the recombinant protein derived from anthracnose dissolved in the solvent is 5 to 30% (w / v), preferably 5 to 20% (w / v).

The crosslinking method using pH can be performed by treating the gel in a basic state, and a buffer solution having a pH of 7 to 12 by adding 10 to 100 mM of sodium hydroxide can be used. In this case, the cross-linking of the protein takes place physically and the gel is easily broken.

In the case of protein bridging using glutaraldehyde, glutaraldehyde is known to play a role in the protein bridge of amine groups, but according to existing literature, various amino acids such as lysine, tyrosine, tryptophan, phenylalanine, histidine, cysteine proline, glycine It is known to be involved in crosslinking of residues.

In the case of gelation of a protein using a photoreactive crosslinking method, an electron acceptor and a photoreactive metal ligand can be added. The electron acceptor is selected from the group consisting of persulfate, periodate, perbromate, perchlorate, vitamin B12, pentaamminechlorocobalt (III), ammonium cerium nitrate but are not limited to, ammonium cerium (IV) nitrate, oxalic acid, or EDTA. The photoreactive metal ligand is selected from the group consisting of ruthenium (Ru), palladium (Pd), copper (II), nickel (II), manganese (Mn) (Fe (III)), but the present invention is not limited thereto.

The method for producing the protein gel is, specifically,

Preparing an expression vector containing a gene of an anemia-derived recombinant protein, preparing a transformant by inserting the expression vector into a host cell,

Producing an anthrax-derived recombinant protein from said transformant,

Separating and purifying the produced anthrax-derived recombinant protein, and

And cross-linking the purified sea anemia-derived recombinant protein.

An embodiment of the present invention can provide a gel comprising a cross-linked product of an anthrax-derived collagen-like recombinant protein and / or an anthrax-derived silk-like recombinant protein.

The hardness of the gel comprising the cross-linked product of the anthrax-derived collagen-like recombinant protein and / or anthrax-derived silk-like recombinant protein may be 0.01 to 3.0 MPa, 0.1 to 3.0 MPa, or 0.05 to 2.5 MPa. In the case of strength, it may be 10 to 1000 kPa or 50 to 900 kPa. The elastic force may be 10 to 150% or 30 to 100%.

An anthrax-derived recombinant protein according to the present invention is a specific protein having high repetition ability. It is capable of producing a large amount of recombinant protein from a transformant and is excellent in mechanical properties such as strength, elasticity and attractive force, Can be usefully used in industrial fields. The preparation can be achieved through gelation of recombinant proteins derived from an anthaxe, and they have mechanical properties and can be utilized as biodegradable protein-based functional medical materials such as cell-supporting fixators, microfilters, enzymes, drug carriers and the like .

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of an expression vector comprising a gene of an anemone-derived collagen-like recombinant protein according to an embodiment of the present invention. FIG.
FIG. 2 is a result obtained by silver staining of an anthrax-derived collagen-like recombinant protein after acid extraction and purification according to an embodiment of the present invention.
FIG. 3 is a result of western blot analysis of the expression level of anthaxanthin-derived collagen-like recombinant protein after acid extraction and purification according to an embodiment of the present invention.
FIG. 4 is a graph showing the results of amino acid composition analysis after acid extraction and purification of an anthrax-derived collagen-like recombinant protein according to an embodiment of the present invention.
FIG. 5A is a graph showing the results of an immuno-histochemical assay using an IgM anti- Nematostella vectensis antibody against an anthrax- derived collagen-like recombinant protein according to an embodiment of the present invention.
FIG. 5B is a graph showing the results of an assay of an antigen antibody reaction through an immunohistochemistry using an anthrax- derived collagen recombinant protein of Nematostella vectensis , an anthrax- derived collagen-like recombinant protein according to an embodiment of the present invention.
6 is a gel-like protein obtained by curing an anthrax-derived collagen-like recombinant protein comprising an amino acid sequence of SEQ ID NO: 35 according to an embodiment of the present invention by photoreaction in a mold and removing the mold.
FIG. 7 shows the tensile strength test results of an anemone-derived collagen-like recombinant protein gel comprising the amino acid sequence of SEQ ID NO: 35 according to an embodiment of the present invention.
FIG. 8 is a schematic diagram of an expression vector containing a gene of an anemone-derived silk-like recombinant protein according to an embodiment of the present invention.
FIG. 9 shows the results of SDS-PAGE analysis of the expression level of the anthrax-derived silk-like recombinant protein after acid extraction and purification according to an embodiment of the present invention.
10 is a result obtained by Western blotting the degree of expression of an anthracnose-derived silk-like recombinant protein after acid extraction purification according to an embodiment of the present invention.
11 is a graph showing the results of amino acid composition analysis of an anthrax-derived silk-like recombinant protein to which an expression-enhancing motif is added.
12 is a gel-like protein obtained by curing an anthrax-derived silk-like recombinant protein consisting of the amino acid sequence of SEQ ID NO: 70 according to an embodiment of the present invention by photoreaction in a mold and removing the mold.
Fig. 13 shows the results of the tensile strength test of an anemone-derived silk-like recombinant protein hydrogel comprising the amino acid sequence of SEQ ID NO: 70 according to an embodiment of the present invention.

The present invention will be described in further detail with reference to the following examples. However, it should be understood that the present invention is not limited to the following examples.

Example  One. Origin of anemone Collagen-like Recombinant protein  Preparation of expression vector

1-1. Origin of anemone Collagen-like Of recombinant protein DNA  Optimization of sequence

The anthrax-derived collagen-like recombinant protein used in this experiment Hypothetical protein, using information from the NCBI Reference Sequence. The ancestral collagen-like recombinant protein was obtained from NCBI (XP_001623116.1), and the amino acid sequence of the aquatic collagen-like protein in nature was doubled.

An anthrax-derived collagen-like recombinant protein predominantly repeats of the amino acid sequence has a biased amino acid sequence because of the repetition of the amino acid sequence, and DNA sequences overlap with each other due to DNA codons mainly used for several amino acids. Such DNA repeat sequences may easily be deleted in E. coli or transcription may occur incompletely. Therefore, it is important to prevent the repetition of the sequence at the DNA level in order to facilitate protein expression. Thus, the sequence was optimized by avoiding repetition of some of the codon sequences of the mRNA sequence of the repetitive sequence recombinant protein.

The optimized anther-derived collagen-like recombinant protein is a protein consisting of a total of 190 amino acids mainly comprising proline (24.6%), leucine (17.4%) and glycine (10% The module has a sequence of unusual structure that is repeated 19 times.

1-2. Origin of anemone Collagen-like Of recombinant protein  Preparation of expression vector

A histidine tag (histidine tag, 6 x his tagged sequence, HHHHHH) was added to the DNA of the anthrax-derived collagen-like recombinant protein optimized in the above example using T7 (pT7) and Nde1 and Xho1 restriction enzymes at both ends, Lt; / RTI > (+). A schematic diagram of an expression vector containing the gene of the recombinant protein prepared above is shown in FIG.

1-3. Origin of anemone Collagen-like The recombinant protein  Production of transformed Escherichia coli to be produced

Escherichia coli TOP10 (Invitrogen), which is commonly used for cloning, and E. coli BL21 (DE3), which is used for protein expression, were reacted using CaCl 2 buffer. Then, the expression vector prepared in Example 1 was transfected into Escherichia coli by a thermal shock method in which the expression vector was allowed to stand at 42 DEG C for 2 minutes.

The Escherichia coli was inoculated into LB medium supplemented with 50 ㎎ / ml ampicillin, and cultured in a 5 l incubator for 12 hours at 37 ° C. to select transformed Escherichia coli.

Example  2. Origin of anemone Collagen-like Of recombinant protein  Expression confirmation and purification

2-1. Of recombinant protein  Expression

To induce the expression of transgenic ancestral collagen-like recombinant protein, IPTG (isopropyl-β-D-thiogalactopyranoside, 1 mM) was added when the absorbance (OD 600 ) of the culture reached 0.6 to 1.0 at 600 nm Respectively.

The cultured cells were centrifuged at 4000 rpm for 10 minutes, the supernatant was removed, and the cells were recovered. The recovered cells were suspended in a lysis buffer (pH 8.0, 50 mM NaH 2 PO 4 , 300 mM NaCl) and then disrupted using an ultrasonic sonicator or a high-pressure homogenizer. In the case of histidine tag separation, a solution containing urea (100 mM NaH 2 PO 4 , 10 mM Tris · Cl, 8 M Urea, pH 8.0) was used as a cell disruption solution.

In order to confirm the expression of collagen-like recombinant protein on an SDS-PAGE, a part of the disrupted cells were divided into a whole cell lysate, a soluble fraction and an insoluble fraction. The remaining whole cell lysate was centrifuged at 9000 rpm for 20 minutes to recover the insoluble fraction.

2-2. Heath Tag The column (His-tag column)  Used Origin of anemone Collagen-like Of recombinant protein  refine

The supernatant recovered in Example 2-1 was separated and purified from an anthrax-derived collagen-like recombinant protein expressed in a water-soluble fraction by using a His tag column.

Proteins from the aqueous fraction were injected into a nickel NTA (nitrilotriacetic acid) column, giving the protein sufficient time to bind to the column. Then, the cleaning solution that is not binding proteins to the column (100 mM NaH 2 PO 4, 10 mM Tris · Cl, 8 M Urea, pH 6.3) to wash, and elution buffer (100 mM NaH 2 PO 4, 10 mM Tris Cl, 8 M Urea, pH 4.5) to separate and purify the protein from the column. The amount of protein isolated using a His tag column is less than 10 mg per liter.

2-3. Heat shock  Used Collagen-like Of recombinant protein  refine

Another method of isolating and purifying collagen-like recombinant proteins derived from an anemone was the thermal shock and acid extraction method.

Specifically, only the insoluble fraction was recovered from the cells disrupted in Example 3-1, washed with a washing solution (50 mM NaH 2 PO 4 , 300 mM NaCl, pH 8.0), and then washed again at about 60 ° C Of thermal shock. In this process, the insoluble fraction was again dissolved in formic acid, dialyzed with water and lyophilized to produce. The production of anthracnose-derived collagen-like recombinant protein obtained by acid extraction is about 50 mg per liter.

The produced sea horse-derived collagen-like recombinant protein comprises the amino acid sequence of SEQ ID NO: 34. Among the anthrax-derived collagen-like recombinant proteins produced from the transformed Escherichia coli, the anthropogenic collagen-like recombinant protein represented by SEQ ID NO: 35 including the histidine tag has a theoretical isoelectric point of 6.69 and a theoretical molecular weight of 41.65 kDa.

Example  3. Refined Origin of anemone Collagen-like Of recombinant protein  Confirm

3-1. silver  Staining method Western Blot

Each of the fractions and the purified sample obtained in Example 3 were diluted in a buffer for SDS-PAGE (1M TrisHCl (pH 6.8), 10% glycerol, 2% SDS, 5% beta-mercaptoethanol, 0.25% bromophenol blue) Diluted and denatured by reacting at 100 DEG C for 5 minutes. Then, the test samples were electrophoresed on a 12% SDS polyacrylamide gel to separate their sizes by molecular weight.

The isolated proteins were confirmed by silver staining or Western blotting. The silver staining method is advantageous in that it is more sensitive than the Coomassie staining method because it stains using the reduction difference of the silver ion binding to the electrophoretic protein functional group. As a result of the silver staining, only the expressed anemia - derived protein was detected, and it was confirmed that the protein was observed in the molecular weight range slightly lower than the theoretical value.

 In the case of Western blotting, the presence of the electrophoresed protein was confirmed using an antigen recognizing the histidine tag of the recombinant protein C-terminal, and the result was also the same as the silver staining method, so that it was confirmed that the target protein could be extracted at a high yield .

The results of silver staining and Western blot analysis of the expression level of collagen-like recombinant proteins derived from an anemone are shown in FIG. 2 and FIG. 3, respectively.

As shown in Fig. 2, the band was confirmed to be around 38 kDa, and the theoretical molecular weight of the heattached protein was 41.65 kDa. However, the molecular weight of the protein was not exactly the same, In the case where the protein is identified at the same position as the silver staining method, the desired target protein appears to be a problem of the expression of the electrophoretic mobility phase. In the case of Western blotting, since the histidine tag at the C-terminus is used, it can be said that the expression was completed to the end of the protein at a normal size.

3-2. Collagen-like Of recombinant protein  Amino acid composition analysis

The amino acid composition of the purified anthrax-derived collagen-like recombinant protein was analyzed in order to accurately confirm whether the expressed anthrax-derived collagen-like recombinant protein is a target protein.

Specifically, an anthrax-derived collagen-like recombinant protein was dissolved in 6N hydrochloric acid and phenol, and the composition of amino acids was analyzed using standard solution amino acids for Hydrolysate Program (H), Sykam.

The result of amino acid composition analysis of an anthrax-derived collagen-like recombinant protein is shown in FIG. 4, and the measured values of amino acid composition analysis results are shown in Table 3.

Amino acid type Measured value Ideal value Asx 10.2 9.5 Thr 1.3 1.1 Ser 5 10 Glx 5.8 4.2 Pro 20.3 24.7 Gly 10.3 10 Ala 3.3 0 Cys 0 0 Val 10 10 Met 0.4 0 Ile 1.5 0.5 Leu 16.3 17.4 Tyr 9.2 10 Phe 3.4 2.6 His 0.8 0 Lys One 0 Arg 1.3 0

The amino acid analysis method is a method of determining the amino acid composition ratio of a protein used in an actual experiment. Therefore, it is possible to measure the approximate purity of the protein used in the experiment by comparing the amino acid composition ratio of the protein with the theoretical amino acid composition ratio of the protein known to the protein sequence. If the two component ratios are different from each other, the protein used in the experiment means that the target protein is not the main target, which means that a relatively large number of miscellaneous proteins exist in the separation or purification process.

As shown in Table 3, when the amino acid composition of the obtained anthrax-derived collagen-like recombinant protein was compared with the amino acid composition of the repeated sequence derived from anchovy, which was confirmed in NCBI, the major amino acids such as proline, glycine, tyrosine and leucine It was confirmed that several composition ratios agree with the values of the recombinant proteins produced in Escherichia coli and the theoretical values.

3-3. Immunohistochemistry  Antibody response test

In the case of an anthrax- derived collagen-like recombinant protein according to the present invention, an antibody against a recombinant protein repeat sequence was prepared , and an anemia ( Nematostella vectensis ) tissues using immunohistochemistry to identify the presence and location of proteins.

Specifically, a paraffin-immobilized anemia sample is cut into 4-μm-thick sections, and paraffin and water are removed. For immunohistochemistry, the primary antibody against the recombinant anomalous recombinant protein is treated and the secondary antibody labeled with HRP avidin is used. Thereafter, DAB is added and waited until the color develops at room temperature. The position of the target protein is confirmed by observing the site where the DAB substrate degraded by the enzyme is deposited.

The resulting anthrax- derived collagen-like recombinant protein comprises the amino acid sequence of SEQ ID NO: 35, and the protein is called Nematostella 5A and 5B show experimental results of immuno-histochemistry using an IgG antibody of vectensis and an anthrax- derived silk protein antibody, respectively.

Example  4. Origin of anemone Collagen-like Of recombinant protein Gelling  exam

4-1. Gel production using photochemical reaction

The crosslinking of the protein obtained in the above examples can also be induced by a photochemical reaction. In particular, the ratio of tyrosine residues in anthrax-derived collagen-like recombinant proteins is as high as 10%, and can induce the formation of di-tyrosine residues through photoreaction. After the protein is dissolved at a high concentration of about 5 to 40% (w / v), the protein is reacted with 1 to 10 mM of Ru (BPY) 3 (Ruthenium-tris (2,2'-bipyridyl) dichloride) mM APS (ammonium persulfate) or SPS (sodium persulfate), and irradiate an LED having a wavelength of 450 nm. The irradiation time can be adjusted according to the concentration of the charged electron donor and the photoreactive metal ligand and the amount of light.

FIG. 6 is a photograph showing a state in which a collagen-like recombinant protein derived from an anemone prepared in Example 2 of the present invention is cured by a photoreaction in a mold.

4-2. Gel  Tensile strength test

The anthrax-derived collagen-like recombinant protein in the form of a thin membrane sheet of 0.5 cm 2 cm obtained in Example 4-1 was subjected to tensile strength test using INSTRON (model name: 3544) strain hardness and toughness, which are the fundamental properties of the material, are obtained by measuring the stress-strain curve.

Stress-strain curve obtained through tensile strength test is an experiment that pulls a thin film sheet type protein specimen until it breaks, and its strength is examined. The strain at this time means elasticity, Stress means strength. The hardness can be expressed by the initial slope of the graph, and the toughness is the energy required to cause the gel to break, which can be determined by the area at the bottom of the graph.

The result of the tensile strength test of the anthrax-derived collagen-like recombinant protein hydrogel obtained after gelation is shown in FIG. 7, and the measured results are shown in Table 4.

burglar
(MPa) Resilience
(%) Hardness
(MPa) tenacity
(MJ / m 3 ) 0.86 (+/- 0.28) 68.31 (+ - 23.89) 2.11 (+/- 0.86) 0.42 (+/- 0.22)

The elasticity of the hydrogel formed from the anthrax-derived collagen-like recombinant protein is about 68% lower than that of elastin (~150%), but better than that of nylon (~30%) or silk (~35%) appear. The hardness, which is an important mechanical property of hydrogels, is about 2.11 Mpa, which is stronger than elastin and synthetic rubber of the same unit area and weaker than the viscid silk of the spider (Araneus series, viscid silk, ~ 3 MPa). In the case of a hydrogel usually made of a chemically synthesized polymer, the hardness and strength are in the range of 1 to 100 kPa, and the protein-based hydrogel is in the lower level. Considering this, it can be seen that the obtained strength and hardness are more improved. Considering that the hardness of the acellular collagen gel is in the range of 2 to 60 kPa, the anthrax-derived collagen-like recombinant protein gel is considered to be a harder gel and thus may be useful for maintaining the three-dimensional structure.

Example  5: Origin of anemone Silk-like Of recombinant protein  Preparation of expression vector

5-1. Origin of anemone Silk-like Of recombinant protein DNA  Optimization of sequence

The anthocyanin silk protein used in this experiment was a hypothetical protein, and the information of the NCBI Reference Sequence (XP_001621085.1) was used.

The ancestral silk protein is a protein consisting of 319 amino acids, which is mainly composed of glycine (35%) and proline (18%), and has 10 amino acids (GPGNTGYPGQ) (SEQ ID NO: 37) And one or two amino acids in the middle or in front of the sequence may be present in a slightly different form [repeated sequence (including modified ones) of anchovy silk protein - repeat sequence consisting of amino acid sequence having the general formula shown in SEQ ID NO: 36 (G / D) (P / S) (G / S) NTG (Y / C) P (G / W) Q (SEQ ID NO: 36)], for example, selected from the group consisting of SEQ ID NOS: 37 to 68 , Preferably GPGNTGYPGQ (SEQ ID NO: 37), DPGNTGYPGQ (SEQ ID NO: 38), GPGNTGCPGQ (SEQ ID NO: 45) or GSGNTGCPGQ (SEQ ID NO: 47)).

The silk-like recombinant protein derived from an anthrax, which is predominantly repetitive of the amino acid sequence, has a biased amino acid sequence because the amino acid sequence repeats mainly, and DNA sequences overlap with each other due to a DNA codon mainly used for several amino acids. In order to prevent the repetition of the sequence at the DNA level and to facilitate the expression of the protein, the codon sequence of some of the mRNA sequences of an anthocyanin silk protein was rearranged and optimized.

In addition, the sequence regulating transcription is largely involved in the formation of the secondary structure of the transcript, which regulates the stop and duration of transcription, so that part of the repeating sequence, which is less likely to form a secondary structure, And to minimize the problems caused by this arrangement.

The amino acid sequence (theoretical pI: 3.98, theoretical molecular weight: 29.35 kDa) of the anthocyanin silk protein present in nature is shown in SEQ ID NO: 69.

5-2. Origin of anemone Silk-like Of recombinant protein  Preparation of expression vector

The DNA of the anthrax-derived silk-like recombinant protein optimized in the above 5-1 was added to restriction enzyme sites of Nde1 and Xho1 at both ends and cloned into the expression vector pET23b (+). The histidine tag was included at the C-terminus of the pET23b (+) vector [6 x his tagged sequence; HHHHHH]. In addition, TrpL motifs were ligated to the N-terminus of anomalous silk proteins using an enterokinase cleavage sequence to increase the expression level of the anthrax-derived silk-like recombinant protein separately (expression enhancement motif sequence; MKAIFVLKDDDDK]. The expression vector prepared above contains the promoter T7 (pT7) for Escherichia coli expression.

A schematic diagram of an expression vector containing the gene of an anthrax-derived silk-like recombinant protein is shown in FIG.

5-3. Origin of anemone Silk-like The recombinant protein  Production of transformed Escherichia coli to be produced

Escherichia coli TOP10 (Invitrogen), which is commonly used for cloning, and E. coli BL21 (DE3), which is used for protein expression, were reacted using CaCl 2 buffer. Then, E. coli was transformed into Escherichia coli by thermal shock method in which the expression vector prepared in Example 5-2 was left at 42 DEG C for 2 minutes to prepare transformed E. coli.

Example  6: Origin of anemone Silk-like Of recombinant protein  Expression confirmation and purification

6-1. Origin of anemone Silk-like Of recombinant protein  Expression

The E. coli transformed in Example 5-3 was inoculated into LB medium supplemented with 50 mg / ml of ampicillin and cultured in a 5 l incubator for 10 hours at 37 ° C with shaking. In order to induce the expression of an anthrax-derived silk-like recombinant protein, IPTG (isopropyl-β-D-thiogalactopyranoside, 1 mM) was added when the absorbance (OD 600 ) of the culture solution reached about 0.8 to 0.9 at 600 nm. The cultured cells were centrifuged at 4000 rpm for 10 minutes, the supernatant was removed, and the cells were recovered. The recovered cells were suspended in lysis buffer (pH 8.0, 50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole) and then disrupted using an ultrasonic sonicator or a high-pressure homogenizer. In order to confirm the expression of recombinant silk proteins derived from an anemone on SDS-PAGE, some of the disrupted cells were divided into whole cell lysate, soluble fraction and insoluble fraction. The remaining whole cell lysate was centrifuged at 9000 rpm for 15 minutes to recover only the supernatant.

6-2. heath -tag The column (His-tag column)  Purification method used

The recovered supernatant was separated and purified from anthracnose - like silk - like recombinant protein expressed in aqueous fraction using a Heath - tag column. The protein in the aqueous fraction was injected into a nickel-NTA (nitrilotriacetic acid) column, giving sufficient time for the protein to bind to the column. Then, the cleaning solution that is not binding proteins to the column (50 mM NaH 2 PO 4, 300 mM NaCl, 30 mM imidazole, pH 8.0) to wash, and elution buffer (50 mM NaH 2 PO 4, 300 mM NaCl, 300 mM imidazole, pH 8.0) to separate the protein from the column and purify it.

6-3. Heat shock and For acid extraction  Refining method

Another method for the isolation and purification of anthocyanin-derived silk-like recombinant proteins was carried out using thermal shock and acid extraction. Specifically, only water-soluble fraction was recovered from the disrupted cells, and thermal shock was further applied thereto at about 80 캜. The aqueous fraction formed in this process was separated by centrifugation, and only the separated aqueous fraction was mixed with formic acid or phosphoric acid. The solution was centrifuged again to remove the insoluble protein reacted with the acid by taking only the supernatant. The obtained supernatant was subjected to dialysis using water for one day to remove components other than water and proteins remaining in the protein aqueous solution. The dialyzed protein was lyophilized to obtain a silk - like recombinant protein derived from anthracnose in powder form.

The amino acid sequence (theoretical pI: 5.11, theoretical molecular weight: 31.88 kDa) of the anthrax-derived silk-like recombinant protein to which the expression-enhancing motif produced from the transformed Escherichia coli was added is shown in SEQ ID NO: 70.

Example  7: Refined Origin of anemone Silk-like Of recombinant protein  Confirm

7-1. SDS - PAGE Wow Western blot  Refined Origin of anemone Silk-like Of recombinant protein  Confirm

Each of the fractions obtained in Example 6 and the purified sample were subjected to SDS-PAGE (0.5 M Tris-HCl (pH 6.8), 10% glycerol, 5% SDS, 5%? -Mercaptoethanol, 0.25% Phenol blue) and then boiled and denatured at 100 DEG C for 5 minutes. These samples were separated by molecular weight by electrophoresis on a 12% SDS-polyacrylamide gel. In addition, expression of silk-like recombinant proteins derived from anthracnose through western blot was confirmed.

The results of SDS-PAGE and Western blot analysis of the expression levels of anthrax-derived silk-like recombinant proteins are shown in FIGS. 9 and 10, respectively.

As shown in FIGS. 9 and 10, an anthrax-derived silk-like recombinant protein has a size of about 30 kDa, but a large amount of protein is found at a position of 60 kDa or more, not 30 kDa, on electrophoresis (SDS-PAGE). It is thought that this phenomenon occurs because of the amino acid sequence of the silk - like recombinant protein derived from anthracnose, which is not properly transferred under electrophoresis or electrophoresis in the form of dimer.

7-2. Refined Origin of anemone Silk-like Of recombinant protein  Amino acid composition analysis

In order to clarify the purification of the anthrax-derived silk-like recombinant protein, amino acid composition analysis of purified anthrax-derived silk-like recombinant protein was performed.

Specifically, purified amino acid-derived silk-like recombinant protein was dissolved in distilled water and amino acid composition analysis was performed using amino acid standard H (Pierce Chem. Co.).

The results of analysis of the amino acid composition of the anthrax-derived silk-like recombinant protein added with the expression-enhancing motif are shown in FIG. 11, and the measured values of the amino acid composition analysis results of the anthrax-derived recombinant silk protein are shown in Table 5 .

Amino acid type Measured value Ideal value Asx 17.2 12.6 Thr 12.3 9.4 Ser 1.8 1.8 Glx 8.4 9.7 Pro 17.6 17.6 Gly 31.7 34.7 Ala 1.8 0.3 Cys 0.25 2.9 Val 0.19 0.3 Met 0.14 0.3 Ile 0.26 0.3 Leu 0.36 0.6 Tyr 5 5 Phe 0 0.3 His 2.1 2.9 Lys 0.84 0.9 Arg 0 0

Example  8. Origin of anemone Silk-like Of recombinant protein Gelling  exam

8-1. Photochemical reaction Origin of anemone Silk-like Of recombinant protein Manufacture of hydrogel

Crosslinking of the anthrax-derived silk-like recombinant protein obtained in the above example can also be induced by photochemical reaction. The ratio of the tyrosine residue of an anthrax-derived silk-like recombinant protein is 5%, which can easily and quickly induce the formation of di-tyrosine residues through photoreaction. After the protein is dissolved at a high concentration of about 10 to 40% (w / v), 1 to 10 mM of Ru (BPY) 3 (Ruthenium-tris (2,2'-bipyridyl) dichloride) mM of ammonium persulfate (APS) or sodium persulfate (SPS), and irradiate an LED having a wavelength of 450 nm. The irradiation time can be adjusted according to the concentration of the charged electron donor and the photoreactive metal ligand and the amount of light. Fig. 12 shows a form in which the recombinant protein is cured by a photoreaction in the mold according to the present embodiment.

8-2. Gel  Tensile strength test

The silk-like recombinant protein derived from anthracnose in the form of a thin film sheet having a shape of 0.5 cm 2 cm obtained in Example 8-1 was subjected to tensile strength test using INSTRON (model name: 3544) strain hardness and toughness, which are the fundamental properties of the material, are obtained by measuring the stress-strain curve.

Stress-strain curve obtained through tensile strength test is an experiment that pulls a thin film sheet type protein specimen until it breaks, and its strength is examined. The strain at this time means elasticity, Stress means strength. The hardness can be expressed by the initial slope of the graph, and the toughness is the energy required to cause the gel to break, which can be determined by the area at the bottom of the graph.

The result of the tensile strength test of the anthrax-derived silk-like recombinant protein hydrogel obtained after gelation is shown in FIG. 13, and the measured results are shown in Table 6.

burglar
(MPa) Resilience
(%) Hardness
(MPa) tenacity
(J / m 3 ) 0.08 (0.02) 41.96 (+ - 9.09) 0.19 (0.02) 19.5 (+ - 3.6)

The hardness of the hydrated gel made of the anthrax-derived silk-like recombinant protein is about 0.08 MPa and the elasticity is about 42%. In the case of a hydrogel made of a chemically synthesized polymer, the hardness and strength are in the range of 1 to 100 kPa, and the protein-based hydrogel is of a lower level. The obtained strength is similar to that of the conventional protein-based hydrogel, It can be seen that the hardness shows an improvement of mechanical properties twice as much. The tissue is most similar because the hardness of the skin is 10 to 100 kPa.

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Composition containing recombinant protein derived from sea          anemone for preparing hydrogel and method for preparing hydrogel          using the same <130> DPP20143733KR <160> 70 <170> Kopatentin 2.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> VARIANT <222> (3) <223> X is Pro or Gln. <220> <221> VARIANT <222> (5) <223> X is Leu or Phe. <220> <221> VARIANT <222> (7) <223> X is Pro or Thr. <220> <221> VARIANT <222> (9) <223> X is Ser or Thr. <220> <221> VARIANT <10> <223> X is Asn or Ile. <400> 1 Gly Leu Xaa Val Xaa Tyr Xaa Pro Xaa Xaa   1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 2 Gly Leu Pro Val Leu Tyr Pro Pro Ser Asn   1 5 10 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 3 Gly Leu Pro Val Leu Tyr Pro Pro Ser Ile   1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 4 Gly Leu Pro Val Leu Tyr Pro Pro Thr Asn   1 5 10 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 5 Gly Leu Pro Val Leu Tyr Pro Pro Thr Ile   1 5 10 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 6 Gly Leu Pro Val Leu Tyr Thr Pro Ser Ile   1 5 10 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 7 Gly Leu Pro Val Leu Tyr Thr Pro Ser Ile   1 5 10 <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 8 Gly Leu Pro Val Leu Tyr Thr Pro Thr Asn   1 5 10 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 9 Gly Leu Pro Val Leu Tyr Thr Pro Thr Ile   1 5 10 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 10 Gly Leu Pro Val Phe Tyr Pro Pro Ser Asn   1 5 10 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 11 Gly Leu Pro Val Phe Tyr Pro Pro Ser Asn   1 5 10 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 12 Gly Leu Pro Val Phe Tyr Pro Pro Thr Asn   1 5 10 <210> 13 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 13 Gly Leu Pro Val Phe Tyr Pro Pro Thr Ile   1 5 10 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 14 Gly Leu Pro Val Phe Tyr Thr Pro Ser Asn   1 5 10 <210> 15 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 15 Gly Leu Pro Val Phe Tyr Thr Pro Ser Ile   1 5 10 <210> 16 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 16 Gly Leu Pro Val Phe Tyr Thr Pro Thr Asn   1 5 10 <210> 17 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 17 Gly Leu Pro Val Phe Tyr Thr Pro Thr Ile   1 5 10 <210> 18 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 18 Gly Leu Gln Val Leu Tyr Pro Pro Ser Asn   1 5 10 <210> 19 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 19 Gly Leu Gln Val Leu Tyr Pro Pro Ser Ile   1 5 10 <210> 20 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 20 Gly Leu Gln Val Leu Tyr Pro Pro Thr Asn   1 5 10 <210> 21 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 21 Gly Leu Gln Val Leu Tyr Pro Pro Thr Ile   1 5 10 <210> 22 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 22 Gly Leu Gln Val Leu Tyr Thr Pro Ser Asn   1 5 10 <210> 23 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 23 Gly Leu Gln Val Leu Tyr Thr Pro Ser Ile   1 5 10 <210> 24 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 24 Gly Leu Gln Val Leu Tyr Thr Pro Thr Asn   1 5 10 <210> 25 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 25 Gly Leu Gln Val Leu Tyr Thr Pro Thr Ile   1 5 10 <210> 26 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 26 Gly Leu Gln Val Phe Tyr Pro Pro Ser Asn   1 5 10 <210> 27 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 27 Gly Leu Gln Val Phe Tyr Pro Pro Ser Ile   1 5 10 <210> 28 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 28 Gly Leu Gln Val Phe Tyr Pro Pro Thr Asn   1 5 10 <210> 29 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 29 Gly Leu Gln Val Phe Tyr Pro Pro Thr Ile   1 5 10 <210> 30 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 30 Gly Leu Gln Val Phe Tyr Thr Pro Ser Asn   1 5 10 <210> 31 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 31 Gly Leu Gln Val Phe Tyr Thr Pro Ser Ile   1 5 10 <210> 32 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 32 Gly Leu Gln Val Phe Tyr Thr Pro Thr Asn   1 5 10 <210> 33 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 33 Gly Leu Gln Val Phe Tyr Thr Pro Thr Ile   1 5 10 <210> 34 <211> 380 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 34 Gly Leu Gln Val Leu Tyr Pro Pro Ser Asn Gly Leu Gln Val Leu Tyr   1 5 10 15 Pro Pro Ser Asn Gly Leu Pro Val Leu Tyr Pro Pro Ser Asn Gly Leu              20 25 30 Pro Val Phe Tyr Pro Pro Ser Asn Gly Leu Pro Val Phe Tyr Pro Ser          35 40 45 Ser Asn Gly Leu Pro Val Phe Tyr Pro Pro Ser Asn Gly Leu Pro Val      50 55 60 Phe Tyr Pro Pro Ser Asn Gly Leu Gln Val Leu Tyr Pro Pro Ser Ile  65 70 75 80 Gly Leu Pro Val Leu Tyr Pro Pro Ser Asn Gly Leu Gln Val Leu Tyr                  85 90 95 Pro Pro Ser Asn Gly Leu Gln Val Leu Tyr Pro Pro Ser Asn Gly Leu             100 105 110 Pro Val Leu Tyr Thr Pro Thr Asn Gly Leu Gln Val Leu Tyr Pro Pro         115 120 125 Ser Asn Gly Leu Gln Val Leu Tyr Pro Ser Ser Asn Gly Leu Pro Val     130 135 140 Leu Tyr Pro Pro Ser Asn Gly Leu Gln Val Leu Tyr Pro Pro Ser Asn 145 150 155 160 Gly Leu Pro Val Phe Tyr Pro Pro Ser Asn Gly Leu Pro Val Leu Tyr                 165 170 175 Pro Pro Ser Asn Gly Leu Pro Val Leu Tyr Pro Pro Ser Asn Gly Leu             180 185 190 Gln Val Leu Tyr Pro Pro Ser Asn Gly Leu Gln Val Leu Tyr Pro Pro         195 200 205 Ser Asn Gly Leu Pro Val Leu Tyr Pro Ser Ser Asn Gly Leu Pro Val     210 215 220 Phe Tyr Pro Pro Ser Asn Gly Leu Pro Val Phe Tyr Pro Ser Ser Asn 225 230 235 240 Gly Leu Pro Val Phe Tyr Pro Pro Ser Asn Gly Leu Pro Val Phe Tyr                 245 250 255 Pro Pro Ser Asn Gly Leu Gln Val Leu Tyr Pro Pro Ser Ile Gly Leu             260 265 270 Pro Val Leu Tyr Pro Pro Ser Asn Gly Leu Gln Val Leu Tyr Pro Pro         275 280 285 Ser Asn Gly Leu Gln Val Leu Tyr Pro Ser Ser Asn Gly Leu Pro Val     290 295 300 Leu Tyr Thr Pro Thr Asn Gly Leu Gln Val Leu Tyr Pro Pro Ser Asn 305 310 315 320 Gly Leu Gln Val Leu Tyr Pro Pro Ser Asn Gly Leu Pro Val Leu Tyr                 325 330 335 Pro Pro Ser Asn Gly Leu Gln Val Leu Tyr Pro Pro Ser Asn Gly Leu             340 345 350 Pro Val Phe Tyr Pro Pro Ser Asn Gly Leu Pro Val Leu Tyr Pro Pro         355 360 365 Ser Asn Gly Leu Pro Val Leu Tyr Pro Ser Ser Asn     370 375 380 <210> 35 <211> 390 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 35 His Met Gly Leu Gln Val Leu Tyr Pro Pro Ser Asn Gly Leu Gln Val   1 5 10 15 Leu Tyr Pro Pro Ser Asn Gly Leu Pro Val Leu Tyr Pro Pro Ser Asn              20 25 30 Gly Leu Pro Val Phe Tyr Pro Pro Ser Asn Gly Leu Pro Val Phe Tyr          35 40 45 Pro Ser Ser Asn Gly Leu Pro Val Phe Tyr Pro Pro Ser Asn Gly Leu      50 55 60 Pro Val Phe Tyr Pro Pro Ser Asn Gly Leu Gln Val Leu Tyr Pro Pro  65 70 75 80 Ser Ile Gly Leu Pro Val Leu Tyr Pro Pro Ser Ser Gn Leu Gln Val                  85 90 95 Leu Tyr Pro Pro Ser Asn Gly Leu Gln Val Leu Tyr Pro Pro Ser Asn             100 105 110 Gly Leu Pro Val Leu Tyr Thr Pro Thr Asn Gly Leu Gln Val Leu Tyr         115 120 125 Pro Pro Ser Asn Gly Leu Gln Val Leu Tyr Pro Pro Ser Asn Gly Leu     130 135 140 Pro Val Leu Tyr Pro Pro Ser Asn Gly Leu Gln Val Leu Tyr Pro Pro 145 150 155 160 Ser Asn Gly Leu Pro Val Phe Tyr Pro Pro Ser Asn Gly Leu Pro Val                 165 170 175 Leu Tyr Pro Pro Ser Asn Gly Leu Pro Val Leu Tyr Pro Pro Ser Asn             180 185 190 Gly Leu Gln Val Leu Tyr Pro Pro Ser Asn Gly Leu Gln Val Leu Tyr         195 200 205 Pro Pro Ser Asn Gly Leu Pro Val Leu Tyr Pro Pro Ser Asn Gly Leu     210 215 220 Pro Val Phe Tyr Pro Pro Ser Asn Gly Leu Pro Val Phe Tyr Pro Ser 225 230 235 240 Ser Asn Gly Leu Pro Val Phe Tyr Pro Pro Ser Asn Gly Leu Pro Val                 245 250 255 Phe Tyr Pro Pro Ser Asn Gly Leu Gln Val Leu Tyr Pro Pro Ser Ile             260 265 270 Gly Leu Pro Val Leu Tyr Pro Pro Ser Asn Gly Leu Gln Val Leu Tyr         275 280 285 Pro Pro Ser Asn Gly Leu Gln Val Leu Tyr Pro Pro Ser Asn Gly Leu     290 295 300 Pro Val Leu Tyr Thr Pro Thr Asn Gly Leu Gln Val Leu Tyr Pro Pro 305 310 315 320 Ser Asn Gly Leu Gln Val Leu Tyr Pro Ser Ser Asn Gly Leu Pro Val                 325 330 335 Leu Tyr Pro Pro Ser Asn Gly Leu Gln Val Leu Tyr Pro Pro Ser Asn             340 345 350 Gly Leu Pro Val Phe Tyr Pro Pro Ser Asn Gly Leu Pro Val Leu Tyr         355 360 365 Pro Pro Ser Asn Gly Leu Pro Val Leu Tyr Pro Pro Ser Asn Leu Glu     370 375 380 His His His His His 385 390 <210> 36 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> VARIANT <222> (1) <223> X is Gly or Asp. <220> <221> VARIANT <222> (2) <223> X is Pro or Ser. <220> <221> VARIANT <222> (3) <223> X is Gly or Ser. <220> <221> VARIANT <222> (7) <223> X is Tyr or Cys. <220> <221> VARIANT <222> (9) <223> X is Gly or Trp. <400> 36 Xaa Xaa Xaa Asn Thr Gly Xaa Pro Xaa Gln   1 5 10 <210> 37 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 37 Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln   1 5 10 <210> 38 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 38 Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln   1 5 10 <210> 39 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 39 Gly Ser Gly Asn Thr Gly Tyr Pro Gly Gln   1 5 10 <210> 40 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 40 Asp Ser Gly Asn Thr Gly Tyr Pro Gly Gln   1 5 10 <210> 41 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 41 Gly Pro Ser Asn Thr Gly Tyr Pro Gly Gln   1 5 10 <210> 42 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 42 Asp Pro Ser Asn Thr Gly Tyr Pro Gly Gln   1 5 10 <210> 43 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 43 Gly Ser Ser Asn Thr Gly Tyr Pro Gly Gln   1 5 10 <210> 44 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 44 Asp Ser Ser Asn Thr Gly Tyr Pro Gly Gln   1 5 10 <210> 45 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 45 Gly Pro Gly Asn Thr Gly Cys Pro Gly Gln   1 5 10 <210> 46 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 46 Asp Pro Gly Asn Thr Gly Cys Pro Gly Gln   1 5 10 <210> 47 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 47 Gly Ser Gly Asn Thr Gly Cys Pro Gly Gln   1 5 10 <210> 48 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 48 Asp Ser Gly Asn Thr Gly Cys Pro Gly Gln   1 5 10 <210> 49 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 49 Gly Pro Ser Asn Thr Gly Cys Pro Gly Gln   1 5 10 <210> 50 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 50 Asp Pro Ser Asn Thr Gly Cys Pro Gly Gln   1 5 10 <210> 51 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 51 Gly Ser Ser Asn Thr Gly Cys Pro Gly Gln   1 5 10 <210> 52 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 52 Asp Ser Ser Asn Thr Gly Cys Pro Gly Gln   1 5 10 <210> 53 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 53 Gly Pro Gly Asn Thr Gly Tyr Pro Trp Gln   1 5 10 <210> 54 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 54 Asp Pro Gly Asn Thr Gly Tyr Pro Trp Gln   1 5 10 <210> 55 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 55 Gly Ser Gly Asn Thr Gly Tyr Pro Trp Gln   1 5 10 <210> 56 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 56 Asp Ser Gly Asn Thr Gly Tyr Pro Trp Gln   1 5 10 <210> 57 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 57 Gly Pro Ser Asn Thr Gly Tyr Pro Trp Gln   1 5 10 <210> 58 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 58 Asp Pro Ser Asn Thr Gly Tyr Pro Trp Gln   1 5 10 <210> 59 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 59 Gly Ser Ser Asn Thr Gly Tyr Pro Trp Gln   1 5 10 <210> 60 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 60 Asp Ser Ser Asn Thr Gly Tyr Pro Trp Gln   1 5 10 <210> 61 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 61 Gly Pro Gly Asn Thr Gly Cys Pro Trp Gln   1 5 10 <210> 62 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 62 Asp Pro Gly Asn Thr Gly Cys Pro Trp Gln   1 5 10 <210> 63 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 63 Gly Ser Gly Asn Thr Gly Cys Pro Trp Gln   1 5 10 <210> 64 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 64 Asp Ser Gly Asn Thr Gly Cys Pro Trp Gln   1 5 10 <210> 65 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 65 Gly Pro Ser Asn Thr Gly Cys Pro Trp Gln   1 5 10 <210> 66 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 66 Asp Pro Ser Asn Thr Gly Cys Pro Trp Gln   1 5 10 <210> 67 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 67 Gly Ser Ser Asn Thr Gly Cys Pro Trp Gln   1 5 10 <210> 68 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 68 Asp Ser Ser Asn Thr Gly Cys Pro Trp Gln   1 5 10 <210> 69 <211> 318 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 69 Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Gly   1 5 10 15 His Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro              20 25 30 Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro          35 40 45 Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn      50 55 60 Thr Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly Gln  65 70 75 80 Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Gly                  85 90 95 Tyr Pro Gly Gln Gly Pro Ser Asn Thr Gly Tyr Pro Trp Gln Gly Pro             100 105 110 Gly Asn Thr Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly         115 120 125 Asn Thr Gly His Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly     130 135 140 Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr 145 150 155 160 Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly Gln Gly                 165 170 175 Ser Gly Asn Thr Gly Cys Pro Gly Gln Gly Ser Gly Asn Thr Gly Cys             180 185 190 Pro Gly Gln Gly Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly         195 200 205 Gln Gly Pro Gly Asn Thr Gly His Pro Gly Gln Gly Pro Gly Asn Thr     210 215 220 Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp 225 230 235 240 Pro Gly Asn Thr Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys                 245 250 255 Pro Gly Gln Gly Ser Gly Asn Thr Gly Cys Pro Gly Gln Gly Ser Gly             260 265 270 Asn Thr Gly Cys Pro Gly Gln Gly Pro Gly Gln Gly Pro Gly Asn Thr         275 280 285 Gly Tyr Pro Gly Gly Gly Gly Pro Ser Asn Thr Gly Tyr Pro Gly Gln Gly     290 295 300 Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr 305 310 315 <210> 70 <211> 339 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 70 Met Lys Ala Ile Phe Val Leu Lys Asp Asp Asp Asp Lys Gly Pro Gly   1 5 10 15 Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Gly His Pro Gly              20 25 30 Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr          35 40 45 Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp      50 55 60 Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys  65 70 75 80 Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly Gln Gly Pro Gly                  85 90 95 Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly             100 105 110 Gln Gly Pro Ser Asn Thr Gly Tyr Pro Trp Gln Gly Pro Gly Asn Thr         115 120 125 Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Gly     130 135 140 His Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro 145 150 155 160 Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr Gly Cys Pro                 165 170 175 Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly Gln Gly Ser Gly Asn             180 185 190 Thr Gly Cys Pro Gly Gln Gly Ser Gly Asn Thr Gly Cys Pro Gly Gln         195 200 205 Gly Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro     210 215 220 Gly Asn Thr Gly His Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro 225 230 235 240 Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn                 245 250 255 Thr Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly Gln             260 265 270 Gly Ser Gly Asn Thr Gly Cys Pro Gly Gln Gly Ser Gly Asn Thr Gly         275 280 285 Cys Pro Gly Gln Gly Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro     290 295 300 Gly Gln Gly Pro Ser Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn 305 310 315 320 Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Leu Glu His His His                 325 330 335 His His His            

Claims (16)

A recombinant protein derived from an anthracnose-derived collagen-like recombinant protein and an anthrax-derived silk-like recombinant protein,
Wherein said anthrax-derived collagen-like recombinant protein comprises a polypeptide in which at least one repeating unit selected from the repeating unit group consisting of the repeating unit having the amino acid sequence of SEQ ID NO: 1 is repeatedly connected by 1 to 200 times, Derived recombinant protein which is linked repeatedly,
Wherein said anthrax-derived silk-like recombinant protein comprises a polypeptide in which at least one repeating unit selected from the repeating unit group consisting of the repeating unit having the amino acid sequence of SEQ ID NO: 36 is repeatedly and repeatedly attached by 1 to 200 times, Wherein the recombinant protein is an anthrax-derived recombinant protein that is repeatedly linked. The composition for gel formation according to claim 1, wherein the repeating unit of the anthrax-derived collagen-like recombinant protein is at least one selected from the group consisting of repeating units consisting of amino acid sequences of SEQ ID NOS: 2, 8, 10, 18 and 19. The composition according to claim 1, wherein the repeating unit of the anthrax-derived silk-like recombinant protein is at least one selected from the group consisting of repeating units consisting of the amino acid sequences of SEQ ID NOS: 37, 38, 45 and 47. The composition for preparing a gel according to claim 1, wherein the polypeptide of the sea anemone-derived collagen-like recombinant protein is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 34 repeatedly and repeatedly from 1 to 20 times. The composition for gel formation according to claim 1, wherein the polypeptide of the anthrax-derived silk-like recombinant protein is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 69, which is repeated 1 to 20 times. The recombinant protein of claim 1, wherein the anthrax-derived recombinant protein comprises a His tag, a GST tag, and a maltose binding protein tag at the C-terminus, N-terminus, or both ends of the protein. Wherein at least one selected from the group is further connected. The composition of claim 1, wherein the anthrax-derived silk-like recombinant protein further comprises an expression-enhancing peptide at the N-terminus of the protein. The composition of claim 1 wherein the composition is selected from the group consisting of phosphoric acid, acetic acid, formic acid, hydrochloric acid, sulfuric acid, nitric acid, citric acid, hexafluoroisopropanol (HFIP), hexafluoropropanol (HFP), hexafluoroacetone Wherein the composition further comprises at least one solvent selected from the group consisting of trifluoroacetic acid (TFA), diisopropylethylamine, and methylimidazolium chloride. The composition of claim 1, wherein the composition further comprises a polymeric compound, an albuminoid, or a mixture thereof. The method of claim 8, wherein the polymer is selected from the group consisting of poly-ethylene oxide (PEG), polyethylene glycol (PEG), polycaprolactone (PCL), polylactic acid (PLA), poly- -L-lactide acid, chitosan, and the albuminolide is at least one selected from the group consisting of keratin, gluten, lactalbumin, collagen, and recombinant proteins thereof. Composition for preparing a gel. 10. A gel production process comprising crosslinking a gel-forming composition according to any one of claims 1 to 10. The gel production method according to claim 11, wherein the crosslinking is carried out by adding at least one crosslinking agent selected from the group consisting of glutaraldehyde, formaldehyde, carbodiimide, and BS3 (bis (sulfosuccinimidyl) suberate) . 12. The method of claim 11, wherein the crosslinking is performed by adding an electron acceptor and a photoreactive metal ligand so that a tyrosine residue contained in the recombinant protein forms a di-tyrosine residue through a photoreaction, Production method. The method of claim 13, wherein the electron acceptor is selected from the group consisting of persulfate, periodate, perbromate, perchlorate, vitamin B12, pentaamminechlorocobalt (III) ), Ammonium cerium (IV) nitrate, oxalic acid, and edetic acid (EDTA). 14. The method of claim 13, wherein the photoreactive metal ligand is selected from the group consisting of ruthenium (Ru), palladium (Pd), copper (II), nickel (Ni) II)), and iron (Fe (III)). A gel comprising a crosslinked product of a protein prepared by crosslinking a composition for making a gel according to any one of claims 1 to 10.
KR1020140102544A 2014-08-08 2014-08-08 Composition comprising recombinant protein derived from sea anemone for preparing hydrogel and method for preparing hydrogel using the same KR20160018234A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110305203A (en) * 2019-07-17 2019-10-08 中国海洋大学 A kind of polypeptide being used to prepare hydrogel
CN116115831A (en) * 2022-10-08 2023-05-16 四川大学 Transdermal photo-curing formed hydrogel with bioactivity and preparation method and application thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110305203A (en) * 2019-07-17 2019-10-08 中国海洋大学 A kind of polypeptide being used to prepare hydrogel
CN110305203B (en) * 2019-07-17 2021-12-10 中国海洋大学 Polypeptide for preparing hydrogel
CN116115831A (en) * 2022-10-08 2023-05-16 四川大学 Transdermal photo-curing formed hydrogel with bioactivity and preparation method and application thereof
CN116115831B (en) * 2022-10-08 2024-02-23 四川大学 Transdermal photo-curing formed hydrogel with bioactivity and preparation method and application thereof
WO2024074120A1 (en) * 2022-10-08 2024-04-11 山西锦波生物医药股份有限公司 Transdermal photocuring forming hydrogel with biological activity, and preparation method therefor and use thereof

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