KR20040015532A - ADHESIVE PROTEIN Mgfp-5 ISOLATED FROM MUSSEL AND METHOD OF PRODUCING SAME - Google Patents

Abstract

PURPOSE: A recombinant adhesive protein Mgfp-5 derived from a mussel and a method for producing the same protein are provided, thereby cheaply mass producing the recombinant adhesive protein and substituting chemical adhesives with the recombinant adhesive protein. CONSTITUTION: A gene mgfp-5(Mytilus galloprovincialis foot protein-5) encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4 is provided, wherein the gene mgfp-5 has the nucleotide sequence of SEQ ID NO: 3. A recombinant adhesive protein Mgfp-5 contains the amino acid sequence of SEQ ID NO: 4. A vector containing the gene mgfp-5 g is provided, wherein the vector is pMDG05(KCTC 10291BP). A transformant transformed with the vector is provided, wherein the transformant is a prokaryote or an eukaryote; and the transformant is E. coli BL21/pMDG05. A method for producing the recombinant adhesive protein Mgfp-5 derived from a mussel comprises the steps of: (a) constructing a vector pMDG05(KCTC 10291BP) containing the gene mgfp-5; (b) transforming a host cell with the vector pMDG05; (c) culturing the transformant to express the recombinant Mgfp-5; and (d) isolating and purifying the recombinant adhesive protein Mgfp-5.

Description

Mussel adhesive protein Mg -5 and its production method {ADHESIVE PROTEIN Mgfp-5 ISOLATED FROM MUSSEL AND METHOD OF PRODUCING SAME}

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to mussel adhesive protein Mgfp-5 and a method for producing the same, more particularly, a novel mgfp-5 gene, a vector comprising the same, a transformant comprising the same, and a recombinant Mgfp-produced from the transformant. It is about five.

[Private Technology]

Mussels, which are marine life, produce and secrete adhesive proteins that allow mussels to attach themselves to wet solid surfaces, such as rocks in the ocean, so they are not affected by waves or buoyancy effects. (JH Waite et al., 1983, Biological Review 58, 209-231).

Mussel adhesive protein is known as a strong natural adhesive when compared with currently known chemical synthetic adhesives, and has the flexibility to bend while showing a tensile strength of about twice as high as most epoxy resins. Mussel adhesive proteins also have the ability to adhere to a variety of surfaces, including plastics, glass, metals, Teflon and biomaterials, and can adhere to wet surfaces in minutes. This property remains an unfinished task in the field of chemical adhesives.

In addition, adhesive proteins are known to not attack human cells or cause immune reactions. Therefore, they are highly applicable to medical fields such as adhesion of living tissues and adhesion of broken teeth during surgery (J. Dove et al., 1986, Journal of American Dental Association 112). , 879).

The mechanism by which mussel adhesive proteins adhere to the surface of substrates is reported by chemical modification of tyrosine residues (TJ Deming, 1999, Curremt Opinion in Chemical Biology 3, 100-105; M. Yu et al., 1999, The Journal of American Chemical Society 121, 5825-5826. In most mussel adhesive proteins, tyrosine accounts for about 20-30% of the total amino acid sequence. Tyrosine in the adhesive protein is added to the OH group through the hydration process to turn into a wave (Dopa), it is assumed that the wave guide plays a major role in adhesion to the surface. In addition, the waveguide is converted into dopa o-quinone through the oxidation process. The dopa quinone plays a role of strengthening adhesion by causing cross-linking between adhesive proteins (JH Waite, 1990, Comparative Biochemistry Physiology Part B 97, 19-27).

Mussel adhesive proteins are currently produced by extraction. However, 10,000 mussels are used to extract 1 g of natural adhesive protein, and a very high price point of about $ 75,000 is formed on 1 g of mussel adhesive protein.

Studies on mussel proteins have been made on Mytilus edulis , Mytilus galloprovincialis and Mytilus coruscus (TJ Deming, 1999, Curremt Opinion in Chemical Biology 3, 100-105).

Until now, research on mussel adhesive protein has been largely divided into the development of various mussel proteins and mass production using genetic recombination technology. The reported mussel adhesive proteins include Mefp (Mytilus edulis foor protein) -1, Mgfp (Mytilus galloprovincialis foor protein) -1, Mcfp (Mytilus coruscus foor protein) -1, Mefp-2, Mefp-3, Mgfp-3, etc. (TJ Deming, 1999, Curremt Opinion in Chemical Biology 3, 100-105; V. Vreeland, 1998, Journal of Phycology 34, 1-8). However, recent studies have found that Mefp-1 is distributed on the surface of mussel adhesive proteins byssus, so that Mefp-1 is not a protein involved in adhesion and is a coating that protects the adhesive in water. (TJ Deming, 1999, Curremt Opinion in Chemical Biology 3, 100-105; LM Rzepecki et al., 1992, Biological Bulletin 183, 123-137), the mussel adhesive protein closest to the substrate to which it adheres. The protein Mefp-5 was found and its nucleotide and amino acid sequences were analyzed (JH Waite et al., 2001, Biochemistry 40, 2887-2893).

On the other hand, Mefp-1 adhesive protein has a nucleotide sequence of about 10 times the decapeptide structure consisting of 10 amino acids, and similar to similar mussel species, Mytileus galofvinialis and Mytileus corpus It has base homology and has a common motif of xYxxxYK in repeated decapeptides (K. Inoue et al., 1996, Journal of Molecular Evolution 43, 348-356). Because of this particular structure, many researchers have attempted to clone proteins that have homology with Mefp-1 or Mgfp-1 and to genetically mass-produce them in recombinant systems such as Escherichia coli, yeast, and insects. In addition, in the case of artificially designed cDNA model peptide that repeats within 10 times to mimic the decapeptide structure attempted to overcome the problem of expression, the expression was successfully confirmed, but the adhesion ability is known to be very insignificant (M. Kitamura et al., 1999, Journal of Polymer Science Part A: Polymer Chemistry 37, 729-736).

In order to solve the problems of the prior art, it is an object of the present invention to provide a novel adhesion protein gene isolated from Mytileus gallobinbinialis.

It is also an object of the present invention to provide a novel adhesive protein isolated from Mytileus galofbinicials.

It is also an object of the present invention to provide a method for producing a large amount of adhesive protein in a biologically active state.

It is another object of the present invention to provide a transformant that efficiently produces adhesive proteins.

It is also an object of the present invention to provide a recombinant adhesive protein produced and separated from the transformant.

Figure 1 is a photograph of the electrophoresis by RT-PCR Mgfp-5 protein cDNA fragment with a template RNA extracted from Mytileus galloprovincial series,

Figure 2 compares the nucleotide sequence of the Mgfp-5 protein of the present invention with the nucleotide sequence of Mefp-5,

Figure 3 compares the amino acid sequence of the Mgfp-5 protein of the present invention with the amino acid sequence of Mefp-5,

4 illustrates a process of preparing pMDG05 vector by inserting mgfp-5 cDNA into pTrcHis vector,

FIG. 5 is a graph showing the growth of recombinant Mgfp-5-producing BL21 Escherichia coli cells according to culture time compared with Escherichia coli transformed with the comparative strain pTrcHis vector,

Figure 6 shows the expression of recombinant Mgfp-5 over time after expression induction by SDS-PAGE (A) and Western blot (B),

7 is a whole cell, cell lysate and cell supernatant prepared to analyze the solubility of recombinant Mgfp-5 expressed in E. coli, and analyzed by SDS-PAGE (A) and Western blot (B),

8 is a chromatograph (A) purified from recombinant Mgfp-5 using affinity chromatography and SDS-PAGE analysis (B) of recombinant Mgfp-5,

9 is a spectral diagram of the amino acid composition analysis of recombinant Mgfp-5 (B) purified from a reference amino acid mixture (A),

10 is a graph measuring the conversion of tyrosine to waveguide and dopaquinone by tyrosinase to confirm chemical modification of recombinant Mgfp-5.

In order to achieve the above object, the present invention provides a mgfp-5 gene encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4.

The present invention also provides Mgfp-5 comprising the amino acid sequence of SEQ ID NO: 4.

The present invention also provides a vector comprising the mgfp-5 gene.

The present invention also provides a transformant transformed with the vector.

The present invention also provides a recombinant Mgfp-5 produced from the transformant.

In another aspect, the present invention (a) preparing a pMDG05 (KCTC 10291BP) vector containing a gene capable of expressing the mgfp-5 gene, (b) transforming the pMDG05 vector to a host cell to prepare a transformant, ( c) culturing the transformant, inducing the expression of recombinant Mgfp-5 to produce recombinant Mgfp-5, and (d) isolating and purifying the recombinant Mgfp-5. Provide a method.

Hereinafter, the present invention will be described in detail.

The present inventors obtained a novel adhesive protein gene from Mytileus galloprovincialis, a type of mussel, and established a system for producing the adhesive protein translated from E. coli.

The present invention provides a mgfp-5 ( Mytilus galloprovincialis foot protein-5) gene encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4. As an example of the mgfp-5 gene, the nucleotide sequence is set forth in SEQ ID NO: 3.

When the mgfp-5 gene of SEQ ID NO: 3 and the Mgfp-5 amino acid sequence of SEQ ID NO: 4 were compared with the previously reported sequence, it was confirmed that the new sequence had not been reported yet.

The present invention also provides a vector comprising a mgfp-5 gene encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4. The vector is capable of expressing mgfp-5, and more preferably, it is a vector capable of expressing Mgfp-5 in prokaryotes or eukaryotes.

Specific examples of the vector may be pGEM-T / mgfp-5 and pMDG05. pGEM-T / mgfp-5 is a cloning vector, mgfp-t is inserted into the Pst I / EcoR I restriction enzyme site of pGEM-T vector, pMDG05 is a vector for E. coli expression and Pst I / EcoR of pTrcHisA vector Mgfp-5 is inserted at the restriction site. The pMDG05 contains a trc promoter, which can induce expression by using isotpropylthio-β-D-galactoside (IPTG), and a histidine affinity ligand for protein separation and purification using affinity chromatography. The genetic sequence for the affinity ligand is present before the mgfp-5 gene. The pMDG05 vector was deposited on June 20, 2002 by the Korea Research Institute of Bioscience and Biotechnology (KCTC) located in Ueun-dong, Yuseong-gu, Daejeon, Korea, and received the accession number KCTC 10291BP.

Also provided is a transformant transformed with the vector of the present invention. The transformant may be prepared by introducing a vector containing the mgfp-5 gene using a prokaryotic-derived cell or a eukaryotic-derived cell as a host. As a specific example, the present invention provides E. coli BL21 / pMDG05.

E. coli BL21 / pMDG05 can be cultured in conventional LB blanks and IPTG can be added to induce expression of the combination Mgfp-5 protein. Preferred recombinant Mgfp-5 expression method is cultured in LB (5 g / liter yeast extract, 10 g / liter Tryptone, 10 g / liter NaCl) medium, the IPTG is 0.1 when the absorbance of the culture is 0.6 to 0.9 at 600 nm 2 to 7 hours of incubation by addition of 10 mM.

The recombinant Mgfp-5 expressed by the above method is produced in water-soluble form, and the cell lysate can be purified by chromatography on a column filled with nickel resin to purify the recombinant Mgfp-5.

The present invention also provides a recombinant Mgfp-5 production method. The production method comprises the steps of (a) preparing a pMDG05 (KCTC 10291BP) vector comprising a gene capable of expressing mgfp-5, (b) transforming the pMDG05 vector to a host cell to prepare a transformant, ( c) culturing the transformant, inducing the expression of recombinant Mgfp-5 to produce recombinant Mgfp-5, and (d) isolating and purifying the recombinant Mgfp-5.

Recombinant Mgfp-5 of the present invention has an adhesive activity can be used for adhesive purposes. Adhesion activity was confirmed by fertilization experiment of tyrosine among the amino acids in recombinant Mgfp-5. Tyrosine is modified into waveguides by tyrosinase, which is transformed into dopaquinones, which are known to act on the surface adhesion of adhesive proteins.

Therefore, the present invention not only cloned the novel mgfp-5 gene, Mgfp-5, but also established a method for producing recombinant Mgfp-5 in a transformant with an adhesive activity. The Mgfp-5 protein and recombinant Mgfp-5 can be used as an adhesive to various substrates, as well as harmless to the human body can be used as a medicine.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Example 1: Cloning of the mgfp-5 Gene

Mussels ( M. galloprovincialis ) were purchased at Jukdo Market in Pohang, stored in ice, transported to a laboratory, and the mussels were cut off. Total RNA from the mussel's foot was isolated by acid guandinium thiocyanate phenol-chloroform method (P. Chomczynski et al., 1987, Analytical Biochemistry 162, 156-159).

Single strand cDNA was synthesized using MLV reverse transcriptase (Gibco-BRL, USA) and oligo-dT 12-18 primer (Gibco-BRL) using the obtained total RNA as a template. The nucleotide sequence to be obtained is determined to be homologous to the recently reported mefp-5 nucleotide sequence (JH Waite et al., 2001, Biochemistry 40, 2887-2893), and as a primer set for PCR, mgfp- Designing and preparing mgfp-5-D of 5-U and SEQ ID NO: 2, PCR was carried out using cDNA as a template to obtain a cDNA having a size of 234 bp, which was named mgfp-5 ( Mytilus galloprovincialis foot protein-5) cDNA (FIG. 2). .

The sequencing of mgfp-5 cDNA was performed in pGEM-T (Promega, USA), a TA cloning vector. As a result of sequencing analysis, the sequence excluding the secretion signal sequence of the gene mgfp-5 is shown in SEQ ID NO: 3, and the amino acid sequence encoded therefrom is shown in SEQ ID NO: 4.

Gene sequence similarity analysis with mefp-5 (Bio-Edit Sequence Alignment Editor, Department of Microiology, Northcarolina State University) was performed, and as a result, mgfp-5 gene was 94% identical to mefp-5 in sequencing comparison (Fig. 2). ), The encoded amino acid sequence is also replaced with alanine (threonine) in the 14th amino acid (lysine and glycine (lysine) and glycine (glycine) was added to the 68th and 72th (Fig. 3) .

Example 2: Vector Construction for Genetic Production of Mgfp-5

The mgfp-5 cDNA contained in the pGEM-T vector was isolated using Pst I and EcoR I restriction enzyme sites, and then inserted into pTrcHisA vector (Invitrogen, USA) cut by Pst I and EcoR I restriction enzymes. pMDG05 (4630bp) was prepared (FIG. 4). pMDG05 was deposited on June 20, 2002 by the Korea Research Institute of Bioscience and Biotechnology (KCTC) located in Ueun-dong, Yuseong-gu, Daejeon, Korea and received the accession number KCTC 10291BP.

pMDG05 vector contains trc promoter, a promoter for E. coli expression, can induce expression using IPTG (isopropylthio-β-D-galactoside, Sigma, USA), and purified by protein separation using affinity chromatography The genetic sequence for histidine affinity ligand for is present in front of the mgfp-5 gene.

Example 3: Preparation of a transformant that produces Mgfp-5

pMDG05 vector was made by using a CaCl ₂ buffer in E. coli BL10 used for cloning E. coli Top10 (Invitrogen) and protein expression, respectively. Transformed by Selection of transformed colonies was performed using ampicillin (Sigma) to obtain E. coli Top10 / pMDG05 and E. coli BL21 / pMDG05.

Example 4: Production of Mgfp-5

(1) Expression of Mgfp-5 from E. coli BL21 / pMDG05

E. coli BL21 / pMDG05 was incubated in LB (5 g / liter yeast extract, 10 g / liter Tryptone, 10 g / liter NaCl) medium, and IPTG was added when the absorbance of the medium reached 0.8 at 600 nm (1). mM) to induce the expression of the recombinant adhesion protein Mgfp-5. At this time, 10 ml of LB medium was added to a 50 ml sterile tube (500 µg of ampicillin) and a culture medium grown for 12 hours was loaded with 100 ml of LB medium. Inoculated into 500 mL flask.

The incubation time the growth of the cells E. coli BL21 / pMDG05 was compared to E. coli BL21 / pTrcHis, expressed as a growth graph in Fig. In FIG. 5, E. coli BL21 / pMDG05 was found to stop growing shortly after induction of expression with IPTG. These phenomena were almost the same, although there were some differences even when cultured at different time points of expression induction.

(2) Measurement of expression pattern of Mgfp-5

In order to analyze the expression of recombinant Mgfp-5 adhesion protein, culture medium was collected after IPTG addition, followed by SDS-PAGE, followed by Western blot.

The whole cell sample from which the supernatant was removed after centrifugation at 4 ° C., 10000 rpm for 10 minutes at 4 ° C. was used for storage and analysis at −80 ° C. every hour. Whole cell samples were diluted in 100 μl of SDS-PAGE buffer (0.5 M Tris-HCl, pH 6.8, 10% glycerol, 5% SDS, 5% β-mercaptoethanol, 0.25% bromophenol blue) and boiled at 100 ° C for 5 minutes. Denatured. In the case of SDS-PAGE, the samples were electrophoresed on 15% SDS-polyacrylamide gel and protein bands were detected using Coomasie blue staining or silver staining (Bio-Rad, USA). In the case of Western blot, the samples were electrophoresed on 15% SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane under 15 V voltage. The protein-transferred nitrocellulose membrane was detected after color reaction using Mgfp-5 protein using monoclonal histidine affinity ligand antibody (R & D Systems, USA).

Figure 6 is an electrophoresis picture (A) and Western blot picture (B) showing the expression pattern with time after expression of recombinant Mgfp-5. Recombinant Mgfp-5 was expressed in a dark band of about 16 kDa size compared to other proteins of Escherichia coli, and its expression amounted to about 10 to 13% of the total protein. Recombinant Mgfp-5 expression was increased after induction of expression and reached a maximum at about 4-5 hours, after which the expression amount was confirmed to be degraded and reduced by protease.

(3) Analysis of Expression Form of Recombinant Mgfp-5

In order to confirm the expression form of recombinant Mgfp-5, the cell samples were divided into whole cell samples and cells (using ultrasonic method), and then divided into cell debris and supernatant, respectively, and subjected to SDS-PAGE and Western blot.

Figure 7 is an electrophoresis picture (A) and Western blotting picture (B) of the cell supernatant and cell debris isolated from E. coli BL21 / pMDG05 culture. In both SDS-PAGE and Western blot, the supernatant of Mgfp-5 was strongly detected in the cell supernatant, whereas only a very small amount was detected in the cell debris, indicating that recombinant Mgfp-5 was expressed in water-soluble form in E. coli. This water-soluble form has the advantage that the protein can be used immediately after separation purification without the need for steps such as refolding (refolding) process for the activity of the adhesion protein.

Example 4: Isolation and Purification of Recombinant Mgfp-5

Affinity chromatography was performed using histidine affinity ligands contained in the pMDG05 vector to isolate and purify recombinant Mgfp-5 expressed in water-soluble form in E. coli BL21 / pMDG05.

E. coli BL21 / pMDG05 was cultured in the same manner as in Example 3, and the expression of recombinant Mgfp-5 was induced. The cultured Escherichia coli cells were centrifuged and then disrupted with buffer (50 mM Tris-HCl, 300 mM NaCl, pH8). .0) was concentrated about 50 times, and then 1 mg / ml lysozyme (lysozyme, Bio Basic Inc., Canada) was added, followed by reaction at 4 ° C. for 20 minutes. Thereafter, using an ultrasonic shredder (sonicator) to 200 W on ice for 10 seconds, and 15 seconds to cool the process by repeating 30 times to complete the cell disruption. The crushed E. coli sample was centrifuged at 14,000 rpm for 20 minutes to remove cell debris, and only the supernatant was separated and used as a sample for purification.

5 mL of Hi Trap ™ Chelating HP (Amersham Bioscience, Sweden) was used as a column for the affinity chromatography, and 5 mL of 0.1 M NiSO ₄ was poured into the column charge. The overall separation and purification was performed using Acta Prime Purification System (Amersham Bioscience). The column was equilibrated with 10 ml of binding buffer (20 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, pH7.4) and 10 ml of supernatant sample was added. The column was sufficiently washed with a binding buffer, and the recombinant Mgfp-5 was isolated and purified while flowing 20 ml of an elution buffer with an imidazole concentration gradient. Over 12 hours dialysis at 4 ℃ in sodium phosphate buffer (pH7.4) to remove a high concentration of imidazole was ^{(dialysis, Spectra / Por ⓡ molecularporous} membrane tubing, Spectrum Lab., USA). Purity and yield of the purified recombinant Mgfp-5 were analyzed by SDS-PAGE using silver staining.

FIG. 8 is an SDS-PAGE photograph (B) for chromatographic analysis of recombinant Mgfp-5 using affinity chromatography (A) and purity of purified Mgfp-5. In FIG. 8, the recombinant Mgfp-5 isolated through affinity chromatography was confirmed to have a purity of about 95%.

Example 5: Amino Acid Analysis of Recombinant Mgfp-5

The amino acid sequence of recombinant Mgfp-5 was analyzed by an amino acid analyzer (Hewlette Packard, USA).

The amino acid sequence was analyzed through a spectrum of amino acids sequentially separated from the Mgfp-5 terminus after obtaining the retention time and quantification criteria of each amino acid from the spectrum of the reference amino acid mixture. Spectrum of sample obtained by treatment with hydrochloric acid (HCl) and high temperature to decompose recombinant Mgfp-5 into amino acids after obtaining the retention time and quantification criteria of each amino acid from the spectrum of reference amino acid mixture (FIG. 11A) The composition of each amino acid residue was analyzed through (FIG. 11B). The analysis results are shown in Table 1 below.

Amino acid Predicted composition (%) Experimental composition (%) (± 30%) Ala (A), Pro (P) 3.39 4.19 Arg (D), Asn (N) 6.78 9.06 Glu (E), Gln (Q) 5.08 5.89 Cys (C) 0.85 - Gly (G) 18.64 26.34 His (H) 9.32 9.23 Iso (I) 0.85 2.15 Leu (L) 2.54 2.35 Lys (K) 14.41 12.44 Met (M) 3.39 3.77 Phe (F) - - Ser (S) 11.02 8.49 Thr (T) 1.69 1.81 Trp (W) 0.85 - Tyr (Y) 16.95 14.28 Val (V) - - Total 100 100

In the case of amino acid composition analysis, it is known that about 30% error occurs due to the loss of amino acid during the decomposition of protein. Therefore, the composition and ratio of amino acid residues expressed through the analysis are encoded from the mgfp- 5 sequence. The results were in close agreement with the composition and ratio of the calculated amino acid residues (Table 1). From this, it was confirmed that the protein expressed and isolated from E. coli BL21 / pMDG05 was Mgfp-5, which is an mussel adhesion protein.

Example 6: Validation of Adhesion Activity of Recombinant Mgfp-5

Among the amino acids of mussel adhesive proteins, tyrosine residues are converted to dopa, and further dopa quinones through chemical modification, and the modified dopa and dopaquinones are known to play an important role in adhesion to surfaces. Chemical modification of recombinant Mgfp-5 was investigated using mushroom-derived tyrosinase enzymes that could mediate these chemical modifications.

25 units / ml of mushroom tyrosinase were mixed with 4.2 μg / ml of recombinant Mgfp-5 and the degree of chemical modification was investigated by changing the absorbance while reacting at room temperature. Formation of the waveguide was measured for absorbance at 280 nm, and formation of the waveguide quinone was measured for absorbance at 395 nm.

FIG. 10 is a graph of absorbance measurements of waveguide and dopaquinone formation by modification of recombinant Mgfp-5. FIG. In FIG. 10, the waveguide was rapidly deformed from tyrosine at the beginning of the reaction, and gradually increased after 3 hours with increasing decrease in absorbance. In addition, since dopa quinone is the next reaction from the tyrosine to the waveguide, the reaction occurs weakly until the reaction time of 2 hours, and then increases rapidly thereafter.

In addition, a small white matter precipitated during the tyrosinase reaction. It is assumed that this is caused by crosslinking between recombinant Mgfp-5s.

As mentioned above, the present invention not only cloned the novel adhesion protein Mgfp-5 but also established a method for mass production thereof. Recombinant Mgfp-5 of the present invention can be produced from a transformant in a water-soluble form, its utilization is easy, can be used as a replacement bioadhesive to replace the existing chemical adhesive, it can be used as a medical adhesive.

Claims (10)

A mgfp-5 ( Mytilus galloprovincialis foot protein-5) gene encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4. The gene according to claim 1, wherein the mgfp-5 gene comprises the nucleotide sequence of SEQ ID NO: 3. Mgfp-5 protein comprising the amino acid sequence of SEQ ID NO: 4. A vector comprising the mgfp-5 gene of claim 1. The vector of claim 4, wherein the vector is pMDG05 (KCTC 10291BP). A transformant transformed with the vector of claim 4. The transformant of claim 6, wherein the transformant is a prokaryotic-derived cell or a eukaryotic-derived cell. The transformant of claim 6, wherein the transformant is E. coli BL21 / pMDG05. Recombinant Mgfp-5 produced from the transformant according to any one of claims 6 to 8. (a) preparing a pMDG05 (KCTC 10291BP) vector comprising the expression of the mgfp-5 gene of claim 1 or 2; (b) preparing a transformant by transforming the pMDG05 vector into a host cell; (c) culturing the transformant and inducing the expression of recombinant Mgfp-5 to produce recombinant Mgfp-5; And (d) isolating and purifying the recombinant Mgfp-5 Mgfp-5 production method comprising a.