The present invention relates to a bioadhesive derived from mussel. In particular, it relates to a novel MGFP-3A MUTANT(Mytilus galloprovincialis foot protein type-3A MUTANT) protein and a recombinant protein that is a hybrid of MGFP-3A MUTANT, FP(Foot Protein)-1 and MGFP-5(Mytilus galloprovincialis foot protein type-5). According to the present invention, the adhesive protein can be economically produced in large scale and can be used instead of chemical adhesives.

Description of the Invention

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The present invention relates to a bio-adhesive derived from mussel, more particularly to a novel Mytilus galloprovincialis foot protein-3A (MGFP-3A) and a recombinant protein that is a hybrid of MGFP-3A, FP (Foot Protein)-1 and MGFP-5 (Mytilus galloprovincialis foot protein-5).

(b) Background of the Invention

Mussels produce and secrete specialized water-resistant bioadhesives, and have been studied as a potential source of water-resistant bioadhesives. They adhere tightly to surfaces underwater using the byssus secreted from the foot of the mussel. At the end of each thread is an adhesive plaque containing a water-resistant glue that enables the plaque to anchor to wet solid surfaces [Waite, J. H., Biology Review. 58:209-231 (1983)]. This strong and water-insoluble adhesion has attracted interest for potential use in biotechnological applications.

In addition, mussel adhesive proteins can also be used as medical adhesives as they are non-toxic to the human body and do not impose immunogenicity [Dove et al., Journal of American Dental Association. 112:879 (1986)]. Moreover, their biodegradable properties make them environmentally friendly.

The byssus can be divided into distal and proximal parts. The proximal part is connected to the stem gland of the mussel foot, while the distal part is connected to the adhesive plaques. The adhesive plaque is composed of five distinct types of proteins: foot protein type 1 (FP-1) to type 5 (FP-5) [Deming, T. J., Current Opinion in Chemical Biology. 3:100-105 (1999)].

All of the mussel adhesive proteins contain high ratios of 3,4-dihydroxyphenyl-L-alanine (DOPA), which is derived from hydroxylation of tyrosine residues [Waite, J. H., Biology Review. 58:209-231 (1983)]. The adhesive proteins closest to the adhesion interface have the highest proportion of DOPA residues [Waite, J. H., Integr. Comp. Biol. 42:1172-1180 (2002)]. In contrast, mussel adhesive protein analogs lacking DOPA show greatly reduced adhesion abilities [Yu et al., Journal of American Chemical Society. 121:5825-5826 (1999)]. Indeed, a biochemical study showed that DOPA residues can enable mussel adhesive protein molecules to cross-link with each other via oxidative conversion to DOPA o-quinone. Thus, the DOPA content of a mussel adhesive protein appears to be specifically related to its adhesive properties.

Currently Cell-Tak, a naturally extracted mussel adhesive protein product, is commercially available. This adhesive is mainly composed of FP-1 and FP-2 type proteins, with a minor portion of FP-3. However, the natural extraction process is labor-intensive and inefficient, requiring around 10,000 mussels for 1 g of protein [Morgan, D., The Scientist. 4:1-6 (1990)].

Therefore, researchers have sought to produce recombinant mussel adhesive proteins, for example FP-1, in expression systems such as Escherichia coli and yeast.

However, these previous studies failed to express functional and economical mussel adhesive proteins due to a number of complications, including a highly biased amino acid composition (5 amino acid types comprise .about.89% of the total amino acids in FP-1), different codon usage preferences between mussel and other expression systems (tRNA utilization problems) and low protein yields [U.S. Pat. No. 5,242,808, Filpula et al., Biotechnol. Prog. 6:171-177 (1990), Salerno et al., Applied Microbiology and Biotechnology 58:209-214 (1993), Kitamura et al., Journal of Polymer Science Part A: Polymer Chemistry, 37:729-736 (1999)].

The inventors of the present invention have separated MGFP-5 encoding a novel adhesion protein from a mussel and established a expression system for the production of an adhesion protein that is more adhesive than a FP-1 and is from a mussel (WO 05/02920) to overcome the problems in the prior art.

However, MGFP-5 has drawback of a low producing yield, and a necessity of improving solubility. And so, the inventors improve yield and solubility of polypeptide by developing the chimeric gene with FP-1 and MGFP-5 (WO 05/02920).

Therefore, an adhesive having excellent physicochemical properties including an adhesion force can be developed by combination with MGFP-5 and existing or novel polypeptide of a mussel.

SUMMARY OF THE INVENTION

To overcome the aforementioned problems in the prior art, an objective of the present invention is to provide a novel adhesive protein derived from a mussel.

Another object of the present invention is to provide a novel recombinant adhesive protein which comprises MGFP-3A MUTANT and at least two mussel adhesive proteins, and/or 6xAKPSYPPTYK and MGFP-5 fused at one or both ends of the MGFP-3A MUTANT.

Further object of the present invention is to provide a novel adhesive protein derived from Mytilus galloprovincialis.

The aforementioned adhesive protein preferably comprises the amino acid sequence shown in SEQ ID NO: 14 and the amino acid sequence shown in SEQ ID NO: 16.

The present invention also provides a recombinant adhesive protein where 6xAKPSYPPTYK or some amino acid sequences derived from MGFP-3A is attached to the carboxyl-terminus and/or amino-terminus of the MGFP-5, mussel adhesive protein.

An example of the aforementioned recombinant adhesive protein is an amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 20, the amino acid sequence shown in SEQ ID NO: 22, and the amino acid sequence shown in SEQ ID NO: 24.

Another objective of the present invention is to provide a novel gene coding a new adhesive protein derived from mussel.

The present invention provides a nucleotide sequence encoding the aforementioned novel adhesive protein derived from M galloprovincialis.

The examples of the aforementioned nucleotide sequence are the nucleotide sequence shown in SEQ ID NO: 13 and the nucleotide sequence shown in SEQ ID NO: 15.

The present invention also provides a recombinant adhesive protein where 6xAKPSYPPTYK or some amino acid sequences derived from MGFP-3A is attached to the carboxyl-terminus and/or amino-terminus of the MGFP-5, mussel adhesive protein.

The present invention provides a polynucleotide encoding the aforementioned recombinant adhesive protein where 6xAKPSYPPTYK or some amino acid sequences derived from MGFP-3A is attached to the carboxyl-terminus and/or amino-terminus of the MGFP-5, mussel adhesive protein.

The examples of the aforementioned nucleotide sequence encoding the above recombinant adhesive proteins are the nucleotide sequence shown in SEQ ID NO: 19, the nucleotide sequence shown in SEQ ID NO: 21, and the nucleotide sequence shown in SEQ ID NO: 23.

The present invention also provides a vector which contains an operably-linked nucleotide sequence encoding an adhesive protein.

The present invention also provides a transformant which contains an operably-linked a nucleotide sequence encoding an adhesive protein.

Another objective of the present invention is to provide a method for producing a mussel adhesive protein in a biologically active form in a large scale.

The present invention also provides a method of producing an adhesive protein which comprises the steps of: (a) constructing a vector which contains an operably-linked nucleotide sequence encoding an adhesive protein; (b) constructing a transformant by transforming a host cell with the aforementioned vector; and (c) producing a recombinant adhesive protein by culturing the aforementioned transformant.

The present invention also provides a method of purifying an adhesive protein which comprises the steps of: (a) lysing the transformant, and then centrifuging it to separate the supernatant and the pellet; (b) making a suspension by adding an acidic organic solvent to the pellet; and (c) centrifuging the suspension to separate the supernatant.

Another objective of the present invention is to provide an adhesive containing an adhesive protein as the active component.

The present invention also provides a method of adjusting the adhesive property of an adhesive comprising controlling the concentration of an adhesive protein which is an active component of the aforementioned adhesive, or treating the adhesive with one or more material selected from the group consisting of oxidant, filler and surfactant.

The present invention also provides a coating material containing an adhesive protein as an active component.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The inventors of the present invention have constructed a gene encoding MGFP-3A MUTANT from a gene encoding an adhesion protein of MGFP-3A of Mytilus galloprovincialis, and have established an E. coli expression system for producing an adhesion protein translated from the gene.

They also established a recombinant adhesion protein that is a fusion protein of three or more mussel adhesion proteins, and a producing system therefore.

The adhesive protein of the present invention has the characteristic of attaching to a wide variety of substrates such as glass, metal, polymer resin, plastic or biological cell membranes such as prokaryotic membranes, eukaryotic membranes, and plant cell walls and lipids.

The adhesive protein of the present invention has at least 50% homology with the amino acid sequence shown in SEQ ID NO: 14, preferably 80%, more preferably 90%, and most preferably at least 95% homology, and at the same time can include amino acid sequences that have adhesive property, for example adhesive property that is similar to the amino acid sequence shown in SEQ ID NO: 14, or amino acid sequences that have 70 to 200% of the adhesive activity of the above.

For example, there is a protein that contains the amino acid sequence shown in SEQ ID NO: 14. An adhesive protein that contains the amino acid sequence as shown in the above SEQ ID NO: 14 is referred to as "MGFP-3A MUTANT" (Mytilus galloprovincialis foot protein type 3A Mutant) from hereon.

A nucleotide encoding MGFP-3A MUTANT can be indicated as a variety of nucleotide sequences depending on the amino acid codon usage, such as the nucleotide sequences shown in SEQ ID NO: 13.

Also, the adhesive protein of the present invention can further contain a peptide at the amino-terminus and/or carboxyl-terminus in order to improve the physicochemical properties of the adhesive protein. The above peptide may be added for the purpose of improving for example, the solubility, adhesion force, degree of crosslinking, and the degree of expression, purification, and recovery of protein. For example, the above peptide can be a general reporter protein such as GST or a histidine tag for the purpose of improving the purification.

The preferred examples of mussel adhesive proteins include MGFP-3A, FP-5, and an amino sequence with homology thereto.

The peptide preferably contains an amino acid sequence derived from an adhesive protein, and more preferably contains an amino acid sequence derived from a mussel adhesive protein. An example of the peptide is the amino acid sequence shown in SEQ ID NO: 25 repeated 1 to 10 times in tandem. In an embodiment of the present invention, a SEQ ID NO: 18 was constructed in which the amino acid sequence shown in SEQ ID NO: 25 is repeated 6 times in tandem, and attached to the amino- and/or carboxyl-terminus of the adhesive protein in the present invention. The amino acid sequence shown in the SEQ ID NO: 25 is a part of the sequence of the FP-1 protein.

Examples of recombinant adhesive proteins further comprising the amino acid sequence as shown in SEQ ID NO:25 are amino acid sequence shown in SEQ ID NO: 22, and amino acid sequence shown in SEQ ID NO:24. In addition, recombinant adhesive proteins including amino acid sequence shown in SEQ ID NO:14, amino acid sequence shown in SEQ ID NO:20, amino acid sequence shown in SEQ ID NO:22, or amino acid sequence shown in SEQ ID NO:24 can include histidine tag sequence attached to carboxyl-terminus and amino-terminus thereof in order to easily purify them. Furthermore, the GST tag can also be used to facilitate the purification and analysis.

The adhesive protein and recombinant adhesive protein of the present invention can be inserted into commonly used expression vectors constructed for expressing exogenous genes, and mass-produced through genetic engineering methods. The above vector may be selected according to the type and characteristics of the host cell used in the production of protein, or it may be newly constructed. Transforming the vector into the host cell and producing the recombinant protein from the transformant can easily be carried out through ordinarily employed methods. Selecting, constructing, transforming the vector and expressing the recombinant protein can be easily carried out by an ordinary person skilled in the art of the present invention, and partial variations in the ordinarily employed methods are also included in the present invention.

The sequence encoding an adhesive protein that is inserted into the vector is a sequence encoding an adhesive protein or a recombinant adhesive protein of the present invention, and is preferably selected from the group consisting of a nucleic acid encoding a protein that has at least 50% homology, preferably 80%, more preferably 90%, and most preferably at least 95% homology with the amino acid sequence shown in SEQ ID NOs: 20, 22, or 24, a nucleic acid encoding a protein that has at least 50% homology, preferably 80%, more preferably 90%, and most preferably at least 95% homology with the amino acid sequence shown in SEQ ID NO: 20, 22, or 24, where 6 histidine residues are additionally attached at the amino-terminus.

In an embodiment of the present invention, MGFP-3A MUTANT sequence shown in SEQ ID NO: 13) was cloned into a pTrcHis C vector and pMDG03 (FIG. 2 (see Original Patent)) is constructed. The nucleotide sequence shown in SEQ ID NO: 17 is a gene encoding 6xAKPSYPPTYK which is 6 tandem repeats of the amino acid sequence shown in SEQ ID NO: 25), and is cloned into pUC18. Afterwards, the MGFP-5 sequence was cloned into a pTrcHisA vector to construct a pMDG05 vector (FIG. 4 (see Original Patent)). Furthermore, in order to construct a vector that expresses a recombinant protein having a structure shown in Table 1 (see Original Patent), the sequences of MGFP-3A MUTANT, SEQ ID NO: 15 and SEQ ID NO: 17 were cloned into a pET-22(b) vector to construct pENG353, pENG153 and pENG 351 vectors (FIGS. 4, 5, 6 (see Original Patent)).

The above pET vector is a widely known vector which contains a T7 promoter, which allows expression of exogenous protein by induction using IPTG(isopropylthio-.beta.-D-galactoside), and which has 6 histidine sequences for protein purification by affinity chromatography at the 5' end of the exogenous gene in order to facilitate protein purification. In the present invention, the pMDG05 vector was deposited at the Korean Collection for Type Cultures (KCTC) at the Biological Resource Center of Korea located at Eouen-dong, Yuseong-gu, Daejon, Republic of Korea as of Jun. 20, 2002, and received an accession number of KCTC 10291BP. The pENG151 vector was deposited as of Jan. 19, 2005 and given an accession number of KCTC 10766BP.

The expression vector for the adhesive protein and recombinant adhesive protein can be transformed into a host cell selected from the group consisting of prokaryotes, eukaryotes, and eukaryote-derived cells, in order to construct a transformant. The prokaryote is selected from the group consisting of E. coli and Bacillus, the eukaryote is selected from the group consisting of yeast, insects, animals, and plants, and the eukaryote-derived cells are plant cells, insect cells, and animal cells, but is not limited thereto.

As an embodiment, pMDG03 was transformed into E. coli BL21 and pENG 353, pENG153 and pENG351 vectors were transformed into E. coli BL21(DE3) to construct E. coli BL21/pMDG03, E. coli BL21(DE3)/pENG 353, E. coli BL21(DE3)/pENG153 and E. coli BL21(DE3)/pENG351. The aforementioned 4 types of transformants can be cultured in typical LB media, and IPTG can be added to induce protein expression. The preferred method of expression of recombinant protein is to culture in LB media (5 g/liter yeast extract, 10 g/liter Tryptone, 10 g/liter NaCl), and adding 0.1 to 10 mM of IPTG when the optical density of the culture solution is 0.6 to 0.9 at 600 nm, then culturing for 2 to 7 hours.

The recombinant protein expressed in the above method is expressed in a water-soluble and/or insoluble form within the transformant, so the isolation and purification depends on how it is expressed. When it is expressed in a water-soluble form, the recombinant protein can be purified by running the disrupted cell supernatant through a chromatography column filled with an affinity resin such as a nickel resin. When it is expressed in a water-insoluble form, the recombinant protein can be purified by suspending the disrupted cell pellet in an acidic organic solvent, preferably an organic solvent with a pH of 1 to 6, then centrifuging the suspension to isolate the upper layer. Examples of the acidic organic solvent are acetic acid, citric acid, and lactic acid, but not limited thereto. The acetic acid used can be 5 to 30 (v/v) %, and preferably the cell pellet is dissolved in 20 to 30 (v/v) % acetic acid solution. The upper layer obtained through treatment with acidic organic solvent can further undergo gel filtration chromatography to further purify the recombinant protein.

According to the method of the present invention, 4 mg/L of the recombinant adhesive protein MGFP-3A MUTANT with at least 95% purity, about 38 mg/L of MGFP-353 of with at least 95% purity, about 36 mg/L of MGFP-153 with at least 95% purity, and about 44 mg/L of MGFP-351 of at least 95% purity can be obtained. While MGFP-353 has an adhesion force three times as high as that of MGFP-3A mutant, and one and a half times as high as MGFP-151, it has low modification efficiency of tyrosine residues due to a low solubility. Thus, MGFP-353 can be used for developing an excellent adhesive protein by improving solubility and concentration.

The adhesive protein and the recombinant adhesive protein obtained through its expression in the present invention have adhesive activity and can be used as adhesives. The adhesive activity was confirmed through the experiment of modifying the tyrosine residues in the protein to 3,4-dihydroxyphenyl-L-alanine (DOPA). Thus, the adhesive protein of the present invention can not only be used as an adhesive for a wide variety of substrates, but also be used as a bioadhesive since it is harmless to the human body.

The present invention also provides an adhesive that contains adhesive protein as an active component. The adhesive protein can be a form where 5 to 100% of its tyrosine residues are modified to DOPA, and the adhesive can additionally contain a substance that modifies the tyrosine residues in the protein to DOPA. A typical example of the above substance is tyrosinase, but is not limited thereto.

The above adhesive can further contain 0.5 to 90% by weight of an excipient that is generally contained in bioadhesives or is pharmaceutically acceptable. Examples of excipients include surfactants, oxidants, and fillers, but are not limited thereto (see: US Pat. Application Publication No. 2003-65060 and U.S. Pat. No. 5,015,677). The surfactant can be cationic, anionic, non-ionic, or amphoteric, where examples are sodium dodecylsulfate and sodium dodecylbenzensulfonate. The oxidant can be selected from the group consisting of tyrosinase, catechol oxidase, glutaraldehyde, formaldehyde, bis(sulfosuccinimidyl) suberate, 3,3'-Dithiobis(sulfosuccinimidyl propionate), O.sub.2, Fe.sup.3+, H2O.sub.2 and IO.sub.4.sup.- (see: Macromolecules 1998, 31, 4739-4745), and the filler can be selected from the group consisting of collagen, hyaluronic acid, condroitan sulfate, elastine, laminin, caseine, hydroxyapatite, albumin, fibronectin, and hybrin.

The adhesive of the present invention can be used to adhere or fix glass, plastic, polymer resin, or biological specimen, and the detailed mode and amount of usage, formulation and other such matters may follow Cell-Tak (BD Biosciences, Two Oak Park, Bedford, Mass., USA) which is currently available commercially. For example, the adhesive of the present invention can be a soluble, water-soluble, or insoluble formulation, and can be used in an amount of 0.01 to 100 ug/cm2 for a substrate but is not limited thereto. Furthermore, the mode of use follows the general mode of adhesive use, and the typical mode is coating.

The aforementioned biological specimen refers to any animal or plant categorized as a biological organism and any part derived from such animal or plant. For example, it refers to cells, tissues, organs, RNA, DNA, protein, peptide, polynucleotide, hormones, and compounds, but is not limited thereto.

Examples of application of the adhesive of the present invention are as follows, but not limited thereto: (1) adhesion of substrates under water (fresh or salt water); (2) orthopedic treatments such as treatment of bone, ligament, tendon, meniscus, and muscle, and implant of artificial materials; (3) treatment of perforations, lacerations, and cuts, and ophthalmic attachments such as corneal implants and artificial corneal implants; (4) dental attachments such as holding retainers, bridges, or crowns in place, securing loose teeth, repairing broken teeth, and holding fillers in place; (5) surgical treatments such as attachment of blood vessels, attachment of cellular tissue, artificial material implants, and closure of wounds; (6) plant attachments such as bonding of transplanted parts and wound healing; (7) drugs, hormones, biological factors, medications, physiological or metabolic monitoring equipment, antibiotics, and cell transplant (see: U.S. Pat. No. 5,015,677).

The present invention also provides a method of adjusting the adhesion force of the above adhesive by treating with a substance selected from the group consisting of surfactant, oxidant, and filler, or controlling the concentration of the adhesive protein which is an active component of the adhesive (see: U.S. Pat. No. 5,015,677). The surfactant, oxidant, and filler are the same as was described above.

The present invention also provides a coating agent which contains the above adhesive protein as an active component. Since the adhesive protein of the present invention has the characteristic of adhering to glass, plastic, polymer resin, or biological specimen, it can not only be used as a coating agent for these substrates, but also coat the surface of substrates that are used underwater to prevent oxidation of the substrates, since the adhesive protein is water-resistant and water-repellent. An example of application of the coating agent is to coat the motor propeller of ships to prevent corrosion, but is not limited thereto.

The above coating agent may consist solely of an adhesion protein, but can additionally contain commonly known adhesives, adhesive proteins other than the adhesive proteins of the present invention, resin contained in commonly known coating agents, organic solvents, surfactants, anticorrosive agents, or pigments. The content of the additional components may be appropriately adjusted within the commonly accepted range depending on the kind of component and formulation of the coating agent. Where an additional component is included, the adhesive protein as an active component is contained in the coating agent at a level that maintains the adhesive activity, and can for example be contained in the coating agent at 0.1 to 80% by weight.

The coating agent of the present invention can be manufactured in the form of cream, aerosol (spray), solid, liquid, or emulsion, but is not limited to these formulations.

Claim 1 of 7 Claims

1. An isolated adhesive protein comprising: a peptide selected from the group consisting of an amino acid sequence as shown in SEQ ID NO:20; an amino acid sequence as shown in SEQ ID NO:22; and an amino acid sequence as shown in SEQ ID NO:24.
 

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