WO2008150101A2 - Chimeric polypeptide including a mussel adheisve protein and extracellular matrix - Google Patents

Abstract

The present invention is related to a chimeric polypeptide comprising a mussel adhesive protein and a biofunctional peptide coupled thereto, a bioadhesive extracellular matrix comprising the chimeric polypeptide, and a method of controlling an adhesiveness of an extracellular matrix.

Description

CHIMERIC POLYPEPTIDE INCLUDING A MUSSEL ADHEISVE PROTEIN

AND EXTRACELLULAR MATRIX

CROSS REFERENCE TO RELATED APPLICATION The present application claims priority to and the benefit of U.S. Ser. No.

60/941,872, filed June 4, 2007 with United States Patent and Trademark Office which is hereby incorporated by reference for all purposes as if fully set forth herein.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention is related to a chimeric polypeptide comprising a mussel adhesive protein and a biofunctional peptide coupled thereto, a bioadhesive extracellular matrix comprising the chimeric polypeptide, and a method of controlling an adhesiveness of an extracellular matrix. The present invention also relates to compositions or bio-coating of said extracellular matrix mimic for life science and medical application with enhanced specific bioactivities and biocompatibility.

Description of the Related Art A naturally occurring extracellular matrix (ECM) is a three-dimensional molecular complex that varies in composition, and includes bioactive components such as laminin, fibronectin, various collagens and other glycoproteins, hyaluronic acid, proteoglycans, and elastins.

The adhesive interaction of cells with their surrounding extracellular matrix (ECM) as it occurs in vivo plays an important role in the organization, homeostasis, migratory properties, growth, differentiation, and other various functions of tissues and organs. Continuous communication between cells and their surrounding ECM environment orchestrates critical processes such as the acquisition and maintenance of differentiated phenotypes during embryogenesis, morphogenesis, angiogenesis, wound healing, and even tumor metastasis. Both biochemical and biophysical signals from the ECM modulate fundamental cellular activities including adhesion, migration, proliferation, differential gene expression, and programmed cell death.

The realization of the importance of cell-ECM interaction in biomedical applications such as medical device or tissue engineering scaffold has led to a renewed interest in developing ECM constituents and/or an artificial ECM to mimic its function in cell culturing and medical applications.

A variety of extracellular matrix materials have been proposed for use in cell culture, tissue engineering scaffold, medical device, and other related applications. Medical grafts and cell culture materials containing submucosa derived from small intestine, stomach or urinary bladder tissue, have been proposed (See e.g., US 5,281,422, US 5,554,389, US 6,099,567, US 6,206,931)

Some extracellular materials are commercially available. For example, SURGISIS®, STRATASISz®, and OASIS® based on small intestinal submucosa are manufactured by Cook Biotech Inc. In addition, Matrigel™ (BD BioSciences), a reconstituted extracellular matrix isolated from the murine Engelbreth-Holm-Swarm (EHS) sarcoma, contains a plethora of growth factors and other biologically active molecules and provides a natural, biocompatible environment to cells, and has widely used for cell culturing or tissue engineering.

All of these extracellular materials are derived from natural basilar membranes, and thus are excellent biocompatible. However, their application has been limited due to lack of defined characteristics. To overcome this defect, many attempts have been made to utilize natural biomaterials or synthetic polymeric materials in order to mimic extracellular matrix with defined physicochemical properties. Unfortunately, cellular interactions with natural or synthetic materials are mediated by nonspecifϊcally adsorbed proteins and are generally difficult to control. Further more, they frequently caused immunogenicity. Therefore, a major challenge for such materials is to generate cell and/or tissue friendly environment to support the complex biological functions of the extracellular matrix (ECM) without immunogenicity. A biologically active peptide such as extracellular matrix (EMC) proteins, oligonucelotide, or the like is useful because the immobilization of said substance on materials provide specificity and biocompatible environment. In order to obtain some of the advantages of natural bioactive compounds such as extracellular matrix (ECM) proteins, a lot of techniques, such as hybrid materials that are primarily composed of standard synthetic polymers but are also grafted or copolymerized with biologically functional segments such as peptides, have been developed. (See, e.g., EP 0 710 666 Al, EP 0 608 095 Bl, EP 1 857545 Al)

More recently, chemically and/or genetically engineered polypeptide or natural protein as a substrate has been adopted. For example, Um^'ted States Patent 6,468,731 disclosed proteins comprising any variety of cell growth and/or healing promoting proteins, such as growth factor. The incorporation of these whole proteins may be designed to provide controlled release thereof in a biological system through further use of enzyme degradation sites.

In addition, a tripartite fusion protein was designed and produced recombinantly. In this study, an engineered bone morphogenetic protein-2 (BMP-2) fusion protein was incorporated into a matrix such as fibrin that was covalently combined with N-terminal transglutaminase substrate (TG) domain to treat cancellous bone autograft. (Schmoekel et al., Biotechnology and Bioengineering, 89, p253-262, 2005). A novel protein substrate for controlling cellular functions was constructed by combining functional units of various proteins where ArgGlyAsp (RGD) sequence functioning as a cell adhesive function, an epidermal growth factor (EGF) as a cell growth function, and a hydrophobic sequence (E 12) as an efficient assembling function, were combined and incorporated into one molecule. (Imen et al., Biomaterials, 27, p3451-3458, 2006).

However, naturally occurring proteins such as collagen may be thermally or enzymatically degraded under physiological conditions while a synthetic ECM is less likely to be degraded. For example, the performance of collagen-based matrix such as ECM mimic depends on the one hand on controlling their functional longevity within growing cells and on the other hand on the preservation of the biological properties of the native collagen component. The functional longevity of the collagen component depends on its capacity to resist specific enzymatic degradation by collagenases (metaloproteinases) secreted by its surrounding cells. This capacity is directly related to the number of intramolecular and intermolecular cross-links within the collagen polymer. The higher the number of cross-links, the higher the resistance to collagenase degradation. For this reason, chemically or photochemically processed collagen has been used as a substrate (see e.g., WO 2006/029571, US 6,682,760, US 6,346,515, US 5,955,438). However, crosslinking of collagen reduces or degrades the normal binding sites seen by other molecules and cells in cell and tissue interactions with the extracellular matrix that surrounds cells. In addition, US 5,958,874 disclosed recombinant fibronectin based extracellular matrix for wound healing, and US 6,331,422 and US 6,607,740 disclosed fibrin based matrix for wound healing. The fibrin matrix was enzymatically modified to incorporate bioactive factors such as cell adhesion peptide or growth factors.

SUMMARY QF THE INVENTION

The aforementioned need is met by the present invention by providing a recombinant mussel adhesive protein-derived composition to mimic an extracellular matrix. Biofunctional peptides are recombinatly incorporated into a recombinant mussel adhesive protein to enhance its specificity and biocompatibility by mimicking ECM functions.

However, the enzymatic incorporation or (photo-)chemical graft of bioactive factors to numerous sites of proteins or synthetic polymer were relatively random process, resulting in non-defined physicochemical properties on the surface. The invention aims at eliminating some of the major disadvantages and limitations of the known techniques described above.

Firstly, it aims at providing a technology that allows to mimic an extracellular matrix in a reproducible way, with almost no limitations in terms of the incorporation of bioactive factors. Secondly, it provides the ability to fabricate patterns with stringent control over the density, distribution and therefore functionality of biochemically or biologically active sites in specific areas of the matrix.

The present invention provides a method for the selective modification of a recombinant mussel adhesive protein in itself, no further requirements to offer biocompatible or biofunctional environment.

The present invention a process that allows for efficient modification of recombinant mussel adhesive protein to produce extracellular matrix mimetics

The present invention also provides a coated surface with said extracellular matrix mimetics for cell and/or tissue culturing or regeneration. The present invention also provides an improved extracellular matrix (ECM) coated surface for proliferating and/or maintaining cells for extended periods of time in culture. In particular, the present invention provides a cell culture product including a substrate; and an ECM mimic coating thereon, wherein the coating is adsorbed or bound to at least one surface of the substrate in a minimal solution concentration sufficient to provide ECM environment on the substrate surface. The total amount of the ECM mimic adsorbed by or bound the substrate surface may range from 1 μg /cm² to 50 μg /cm².

BRIEF DESCRIPTION OF THE DRAWINGS These and other features and advantages of the present invention are evident from the following embodiments when read in conjunction with the accompanying drawings in which:

FIG. 1 illustrates general approach to construct cloning vectors to product mussel adhesive protein recombinantly functionalized with biofunctional peptides FIG. 2 illustrates expression and purification of fp-151 -motifs as bioartificial mimics of ECMs including fibronectin, laminin, collagen, and growth factor. (A) Expression and purification of fp-151-GRGDSP, (B) MALDI-TOF MS analysis of purified fp-151-GRGDSP, and (C) expression and purification of other fp-151-ECM peptides FIG. 3 illustrates AFM topographies of (A) uncoated surface, (B) unmodified φ-151 -coated surface, (C) modified fp-151 -coated surface, (D) unmodified fp-151- GRGDSP-coated surface, and (E) modified fρ-151-GRGDSP-coated surface. All topographies were taken in tapping mode under dry conditions.

FIG. 4 illustrates adhesion assay of (A) human HeLa, (B) human 293T, and (C) hamster CHO cells on uncoated (NC), PLL-, Cell-Tak-, fp-151-, and fp-151- GRGDSP-coated polystyrene surfaces.

FIG. 5 illustrates spreading assay of NIH/3T3 cells on (A) uncoated (NC), (B) fp-151-, (C) fp-151 -GRGDSP-, (D) fp-151-IKVAV-, (E) fp-151-YIGSR-, (F) fp- 151-collagenlV-, (G) Cell-Tak-, and (H) PLL-coated polystyrene surfaces. FIG. 6 illustrates proliferation assay of (A) NIH/3T3, and (B) human 293T cells on uncoated, fp-151-, fp-151 -GRGDSP-, fp-151-IKVAV-, fp-151-YIGSR-, fp- 151-collagen IV, Cell-Tak-, and PLL-coated polystyrene surfaces.

FIG. 7 illustrates proliferation assay of MC3T3-E1 cells on uncoated, fp-151-, fp-151-GRGDSP-, fp-151-IKVAV-, fp-151-YIGSR-, fp-151 -collagen IV, Cell-Tak-, PLL-coated polystyrene surfaces.

FIG. 8 illustrates migration activity analysis of human umbilical vein endothelial cells from non-, fp-151-NFG-, and connective tissue growth factor- treatments.

FIG. 9 illustrates tube formation analysis of human umbilical vein endothelial cells from non-, fp-151-NFG-, and naturally occurring angiogenic factor- treatments.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention provides an extracellular matrix mimic for life science and medical applications comprising a recombinant mussel adhesive protein, wherein the recombinant mussel protein is genetically engineered with numerous biologically active polypeptides.

As used herein, the term "biofunctional peptide" refers to a biologically active polypeptide or oligopeptide that play a biological role by acting at specific receptor and/or binding sites at different locations in the cells, tissues, or organism. The term "biological role" refers to the control of biological responses of a cell adhered thereto and/or of a cell in the vicinity of cells adhered thereto. More particularly, the biological response of a cell (adhered to the biofunctional peptide or in the vicinity of the biofunctional peptide) relates to its ability to adhere to a specific substrate, to migrate on this specific substrate, to grow and divide, to grow into a differentiated cell, to express differentiation markers, to form differentiated structures, to respond to a biological stimulus, to communicate with neighboring cells, and/or to organize its cytoskeleton with respect to other cells or with respect to one of the axis of the biofunctional peptide, to express different sets of genes, to express different proteins, to bear different lipids or carbohydrate structure, to adopt different phenotypes, etc.

The term "peptide" includes all moieties containing one or more amino acids linked by a peptide bond. In addition, this term includes within its ambit polymers of modified amino acids, including amino acids which have been post-translationally modified, for example by chemical modification including but not restricted to glycosylation, phosphorylation, acetylation and/or sulphation reactions that effectively alter the basic peptide backbone. Accordingly a peptide may be derived from a naturally-occurring protein, and in particular may be derived from a full- length protein by chemical or enzymatic cleavage, using reagents such as CNBr, or proteases such as trypsin or chymotrypsin, amongst others. Alternatively, such peptides may be derived by chemical synthesis using well known peptide synthetic methods. Included in the scope of the definition of the term "peptide" is a peptide whose biological activity is predictable as a result of its amino acid sequence corresponding to a functional domain. Also encompassed by the term "peptide" is a peptide whose biological activity could have been predicted by the analysis of its amino acid sequence.

The present invention is not limited by the source of the peptide, and clearly extends to peptides and peptide mimetic which are derived from a natural occurring or a non-natural source. The term "derived from" shall be taken to indicate that a particular peptide or mixture of peptides which has been obtained from a particular protein, protein mixture or protein-containing biological extract, either directly (for example, by proteolytic, chemical or physical digestion of the protein(s) or extract) or indirectly, for example, by chemical synthesis of peptides having amino acid sequences corresponding to naturally-occurring sequences, or peptide variants thereof.

A peptide "derived from" a polypeptide having a particular amino acid sequence is any molecular entity which is identical, substantially homologous, or otherwise functionally or structurally equivalent to that polypeptide. Thus, a molecule derived from a particular polypeptide may encompass the amino acid sequence of the polypeptide, any portion of that polypeptide, or other molecular entity that functions to regulate cell-ECM communications. A molecule derived from such a binding domain will mimic the polypeptide from which it is derived. Such molecular entities may include peptide mimetics and the like.

The term "peptide mimetics", as used herein, include synthetic structures which may or may not contain amino acids and/or peptide bonds, but retain structural and functional features of a peptide from which they are derived. The term, "peptide mimetics" also includes peptoid and oligopeptoids, which are peptides or oligomers of N-substituted amino acids (Simon et al., Proc. Natl. Acad. Sci USA, 89, p9367- 9371, 1972). Further included as peptide mimetics are peptide libraries, which are collections of peptides designed to be a given amino acid length and representing all conceivable sequences of amino acids corresponding thereto.

The term "polypeptide" refers to a polymer of amino acids and does not limit the size to a specific length of the product. However, as used herein, a polypeptide is generally longer than a peptide and may include one or more copies of a peptide of interest. This term also optionally includes post expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogues of an amino acid or labeled amino acids.

The term "extracellular matrix (ECM)" refers to a substrate and/or scaffold in the cell's external environment with which the cells can interact via specific cell surface receptors or binding sites.

The term "ECM proteins" refers to fibrous proteins including fibronectin, laminin, vitronectin, collagen, and growth factors, whether naturally occurring or synthetic analogs, as long as it is biologically active. The term "ECM protein segment" refers to any active analogs, fragments or derivatives of ECM proteins.

The term "genetically engineered or recombinantly incorporated" refers to the direct manipulation of an organisms genes via genetic introduction and/or manipulation of DNA in the form of a gene which in turn finds expression to produce favorable and/or desirable physical or biofunctional characteristics of a protein.

The term "progenitor cell" refers to a stem cell with more specialization and less differentiation potential than a totipotent stem cell. For example, progenitor cells include unipotential cells such as fibroblast or osteoblast.

The term growth factor refers to a naturally occurring protein capable of stimulating cellular proliferation and cellular differentiation. Growth factors are important for regulating a variety of cellular processes.

Growth factors typically act as signaling molecules between cells. Examples are cytokines and hormones that bind to specific receptors on the surface of their target cells. They often promote cell differentiation and maturation, which varies between growth factors. For example, bone morphogenic proteins stimulate bone cell differentiation, while fibroblast growth factors and vascular endothelial growth factors stimulate blood vessel differentiation.

The term "growth factor mimetic" includes any active analogs, fragments or derivatives of natural growth factors such as NGF, FGF, PDGF, IGF, BDGF, and substance P.

In an embodiment, the present invention provides a chimeric polypeptide comprising a mussel adhesive protein and a biofunctional peptide coupled to the mussel adhesive protein. Tthe biofunctional peptide is linked to C-terminus, N- terminus or C- and N-terminus of the mussel adhesive protein. The chimeric polypeptide further comprises a space linker peptide in length of 2 to 10 amino acids. The spacer linker peptide is selected from the group consisting of peptides comprising amino acid sequence of SEQ ID NOs: 27 to 29.

The mussel adhesive protein is a fusion protein comprising a first peptide of mussel foot protein (FP)-5 and a second peptide of at least one selected from the group consisting of mussel FP-I, mussel FP -2, mussel FP-3, mussel FP-4, mussel FP-6 and fragment thereof, and the second peptide is linked to C-terminus, N- terminus or C- and N-terminus of the FP-5. The FP-5 comprises SEQ ID NO: 30. The mussel adhesive protein comprises FP-5 and FP-I, or FP-5 and FP-3. The FP-I comprises an amino acid sequence of SEQ ID NO: 32 tandemly repeated 1 to 80 times. The FP-3 comprises SEQ ID NO: 31.

The biofunctional peptide is derived from extracellular matrix protein. The extracellular matrix protein is fibronectin, laminin, collagen, vitronectin, substance P, or nerve growth factor. The biofunctional peptide is derived from a cell binding domain or heparin binding domain of fibronectin. The biofunctional peptide is selected from the group consisting of peptides comprising an amino acid sequence of SEQ ID NOs: 1 to 11. The biofunctional peptide is a peptide having an amino acid sequence of SEQ ID NO: 2. The examples of cell binding domain of fibronectin are Arg GIy Asp (SEQ ID NO:1), GIy Arg GIy Asp Ser Pro(GRGDSP)(SEQ ID NO:2), Tyr GIu Lys Pro GIy Ser Pro Pro Arg GIu VaI VaI Pro Arg Pro Arg Pro GIy VaI (YEKPGSPPREVVPRPRPGV)(SEQ ID NO:3), and Lys Asn Asn GIn Lys Ser GIu Pro Leu lie GIy Arg Lys Lys Thr Asp GIu Leu (KNNQKSEPLIGRKKTDEL) (SEQ ID NO:4), but not limited thereto. The examples of heparin binding domain of fibronectin are Lys Asn Asn GIn Lys Ser GIu Pro Leu lie GIy Arg Lys Lys Thr (KNNQKSEPLIGRKKT)(SEQ ID NO:5), Tyr Arg VaI Arg VaI Thr Pro Lys GIu Lys Thr GIy Pro Met Lys GIu (YRVRVTPKEKTGPMKE) (SEQ ID NO:6), Lys Asn Asn GIn Lys Ser GIu Pro (KNNQKSEP) (SEQ ID NO:7), Lys Ser GIu Pro Leu lie GIy Arg (KSEPLIGR) (SEQ ID NO:8), Ser Pro Pro Arg Arg Ala Arg VaI Thr (SPPRRARVT) (SEQ ID NO:9), Trp GIn Pro Pro Arg Ala Arg He (WQPPRARI)(SEQ ID NO: 10), and Tyr Ala VaI Thr GIy Arg GIy Asp Ser Pro Ala Ser Ser Lys Pro He Ser He Asn Tyr Arg Thr GIu He Asp Lys Pro Ser GIn Met (SEQ ID NO: 11), but not limited thereto.

The biofunctional peptide is derived from laminin. The biofunctional peptide is selected from the group consisting of peptides comprising an amino acid sequence of SEQ ID NOs: 12 to 18. The biofunctional peptide is a peptide having an amino acid sequence of SEQ ID NO: 12 (Tyr He GIy Ser Arg) or a peptide having an amino acid sequence of SEQ ID NO: 17 (He Lys VaI Ala VaI).

The biofunctional peptide is derived from collagen type I or collagen type IV. The biofunctional peptide is a collagen-derived peptide selected from the group consisting of peptides comprising an amino acid sequence of SEQ ID NOs:19 to 21.

The biofunctional peptide is a vitronectin-derived peptide comprising an amino acid sequence of SEQ ID NO:22, a substance P-derived peptide comprising an amino acid sequence of SEQ ID NO:23, or a nerve growth factor-derived peptide selected from the group consisting of peptides comprising an amino acid sequence of

SEQ ID NOs:24 to 26.

In another embodiment, the present invention provides a bioadhesive extracellular matrix comprising the chimeric polypeptide which comprises a mussel adhesive protein comprising and a peptide derived from an extracellular matrix protein coupled to the mussel adhesive protein.

Mussel adhesive protein is a water-resistant bioadhesive, and has been studied as a potential source of medical materials such as surgical sealant as well as tissue engineering scaffolds as they are non-toxic to the human body and do not impose immunogenicity (Dove et al., Journal of American Dental Association, 112, p879, 1986). Moreover, mussel adhesive protein is not enzymatically degradable under cell and/or tissue culture conditions even if its biodegradability can be controllable.

We recently engineered E. coli bacteria to produce a variant of the mussel adhesive proteins in an efficient way (see Cha et al., Biotechnology Journal, 3, p631- 638, 2008, WO 2005/092920 or WO 2006/107183), and the proteins are commercially available under Trademarks MAPTrix™ marketed by Kollodis BioSciences, Inc. Unlike a natural protein that is used for extracellular matrix mimics, this method provides a recombinant expression system for large scale production of mussel adhesive protein with amazingly low cost and adhesiveness. Therefore, the recombinant mussel adhesives serve as good candidate material to design an ECM mimic. The preferred mussel adhesive proteins are specifically described in WO 2005/092920, and WO2006/107183A1 which are hereby incorporated by reference for all purposes as if fully set forth herein.

The extracellular matrix (ECM) mimic of the present invention comprises a recombinant mussel protein, wherein the recombinant mussel protein is genetically engineered with one or two biofunctional peptides. These components are necessary for present invention to mimic the functions of a natural extracellular matrix. Additional components such as growth factors, for example, nerve growth factor or substance P, may also be included to further enhance the beneficial effect of the ECM mimic on cell and tissue culture, medical device and treatment, or any other related application.

Biofunctional peptides are naturally or synthetically derived from ECM proteins to enhance biological functions and biocompatibility of the ECM mimic. The ECM proteins can be fibrous proteins such as collagens, fibronectin, laminin, vitronectin, and the like. ECM proteins can influence integrin activity, and in turn, integrins may activate signaling pathways by coclustering with kinases and adaptor proteins in focal adhesion complexes after their association with polyvalent extracellular matrix (ECM) proteins. For example, a RGD containing peptide segment from fibronectin, laminin or vitronectin to integrins may regulate to its integrin activity.

Suitable peptide fragment of ECM proteins that together forms the ECM mimic are selected from fibronectin. Firbronectin is a multi-domain, multifunctional cell adhesion protein found in blood and in a variety of tissue extracellular matrices (Yamada et al., J. Plast Reconstr Hand Sug. 29, ρ211-219, 1996). Its functional domain includes two fibrin binding domain; a collagen or gelatin binding domain and a cell binding domain; and two heparin binding domains. The peptides from one or more fibronectin domains are selected from the domains described above. Suitable peptides include those from the cell binding domain such as peptides including the amino acid sequence SEQ ID NO:1 to 4, and the heparin binding domain such as the peptides including the amino acid sequence SEQ ID NO: 5 to 10

The selected the peptides from one or two fibronectin domains will be recombinantly incorporated into a mussel adhesive protein to produce an ECM mimic.

Preferably, the peptide fragment derived from fibronectin is a RGD containing peptide.

More preferably, the RGD containing peptide of which the amino acid sequence is GRGDPS (SEQ ID NO: 2).

Laminin is a component of the extracellular matrix that is able to promote neuron attachment and differentiation, in addition to axon growth guidance. Laminin binds to type IV collagen, heparin, gangliosides, and cell surface receptors and promotes the adhesion and growth of various epithelial and tumor cells as well as neurite outgrowth. Laminin is thought to mediate cell-matrix interactions and to be a structural component of all basement membranes binding to collagen IV, heparan sulfate proteoglycan, and nidogen-entactin. Laminin has also been shown to influence the metastatic behavior of tumor cells (see, U.S. Pat. No. 5,175,251).

Active domains have been localized in laminin, based on recent progress in cloning the laminin chains. The Bl chain comprises some 1786 amino acids which appear to form at least six contiguous structural domains. Domains III and V contain homologous repeats rich in cysteine, and could form rather rigid structures adjacent to the globules formed by domains IV and VI. A sequence of some five to nine amino acids in domain III is at least partly is known for responsible for the cell attachment, chemotactic, and receptor binding activities of laminin. This sequence also may have antimetastatic activity with tumor cells (Yamada et al., U.S. Pat. No.5,092,885). The peptides from one or more domains III are selected from the domains described above. Suitable peptides include those derived from the domain III such as peptides including the amino acid sequence SEQ ID NOs: 11 to 15.

Preferably, the peptide fragment derived from laminin is a YIGSR containing peptide. More preferably, the YIGSR containing peptide of which the amino acid sequence is CDPGYIGSR (SEQ ID NO: 15) or YIGSR (SEQ ID NO: 12). Another core functional site for bioactivity in laminin is its core protein domain He Lys VaI Ala VaI (IKVAV)(SEQ ID NO: 17), which is located in the alpha-1 chain of laminin (Shin et al., Biomaterials, 24, p4353-5364, 2003). Laminin is known to stimulate neurite outgrowth and it plays a role in the developing nervous system. It is known that gradients are critical for the guidance of growth cones to their target tissues in the developing nervous system. The peptides derived from one or more domains III are selected from the domains described above. Suitable peptides include those from the domain III such as peptides including the amino acid sequence SEQ ID NO:17 to 18.

Preferably, the peptide fragment derived from laminin is a IKVAV containing peptide. More preferably, the IKVAV containing peptide of which the amino acid sequence is CSRARKQ AASIKVAVSADR (SEQ ID NO: 18), or IKVAV (SEQ ID NO: 17).

Collagens are the most abundant proteins found in the animal kingdom, and are the major proteins comprising an extracellular matrix. There are at least 12 types of collagen. Types I, II, III, and IV collagen are among the most important structural proteins.

Type I collagen, in particular, is involved in a variety of physiological processes through interactions with various cells and tissues. The αl chain of type I collagen has been shown to be associated with interactions of a wide variety of cell types. Suitable peptide derived from particular fragments of the triple-helical domain of the αl chain of type I collagen are selected as biofunctional peptide. Such peptides include those from the domain III such as peptides including the amino acid sequence SEQ ID NO: 19.

Preferably, the peptide fragment derived from collagen I is a primarily repeating GIy-X-Y triplets, which induces each chain to adopt a left-handed poly- triple-helical conformation. More preferably, the peptide fragment is GIy Pro Lys GIy AIaAIa GIy GIu Pro GIy Lys Pro (SEQ ID NO:19).

Type IV collagen forms a two-dimensional reticulum and is a major component of the basal lamina. Previous studies have shown that collagen type IV has a crucial role in the early stage of differentiation of F9 stem cells (Watanabe et al., Pathobiology, 70, p219-228, 2002; Yamashita et al., Nature, 408, p92-96, 2000).

Type IV collagen is a distinctive glycoprotein which occurs almost exclusively in basement membranes, structures which are found in the basal surface of many cell types, including vascular endothelial cells, epithelial cells, etc. Type IV collagen is a major component of basement membranes. It differs from interstitial collagens. Type IV collagen consists of three polypeptide chains: two αl chains and one α2 chain. Type IV collagen has two major proteolytic domains: a large, globular, non-collagenous, NCl domain and another major triple-helical collagenous domain. The latter domain is interrupted by non-collagenous sequences of variable length. The peptide segment, GIy VaI Lys GIy Asp Lys GIy Asn Pro GIy Trp Pro GIy Ala Pro (GVKGDKGNPGWPGAP) (SEQ ID NO: 20), from the αl chain of type IV collagen found to promote the adhesion and spreading of many cell types, and was a potent attractant for melanoma cell motility, (see. U.S. Pat. No.5,082,926)

Vitronectin is an abundant glycoprotein found in blood plasma and the extracellular matrix. Vitronectin has been speculated to be involved in hemostasis and tumor malignancy. The protein consists of three domains: The N-terminal Somatomedin B domain (1-39), A central domains with hemopexin homology (131- 342), and A C-terminal domain (residues 347-459) also with hemopexin homology. The Somatomedin B domain of vitronectin binds to plasminogen activator mhibitor- 1 (PAI-I), and stabilizes it. Thus vitronectin serves to regulate proteolysis initiated by plasminogen activation. Additionally vitronectin is a component of platelets and is thus involved in hemostasis. Vitronectin contains an RGD (45-47) sequence which is a binding site for membrane bound integrins, e.g. the vitronectin receptor, which serve to anchor cells to the extracellular matrix. Preferably, the fragment is derived from Somatomedin B domain of which the amino acid sequence is Lys Lys GIn Arg Phe Arg His Arg Asn Arg Lys GIy Tyr Arg Ser GIn(KKQRFRHRNRKGYRSQ) (SEQ ID NO: 22).

In one embodiment of the invention, a method of preparing an ECM mimic is provided. The method comprises the steps of the construction of recombinant plasmid pFP151-ECM derived polypeptide or oligopeptide that can express fusion protein of hybrid fp-151 and a peptide derived from the peptide of which the amino acid sequence is selected from the group consisting of RGD-containing fragment such as GRGDSP (SEQ ID NO:2), can be constructed. Obviously ECM derived polypeptide or oligopeptide can be from any sources. Preferred fibronectin-, laminin-, collagen-, vitronectin-, heparin binding domain-derived peptide are GRGDSP(SEQ ID NO:2), YIGSR(SEQ ID NO:12) and/or IKAVA(SEQ ID NO: 17), GIy GIu Phe Tyr Phe Asp Leu Arg Leu Lys GIy Asp Lys (GEFYFDLRLKGDK)(SEQ ID NO: 21), and KNNQKSEP(SEQ ID NO: 7), respectively.

The role of nanoscale organization of RGD peptides in the regulation of the adhesion, proliferation and differentiation of both preosteoblasts (MC3T3-E1) and multipotential (Dl) cell lines in vitro indicated increased RGD island (defined as a cluster of RGD peptides) spacing on alginate substrate was observed to promote spreading of MC3T3-E1 cells while simultaneously suppressing their proliferation. However, increased RGD island spacing decreased spreading of Dl cells while also decreasing proliferation. Moreover, differentiation of preosteoblasts was significantly upregulated in response to decreased RGD island spacing, whereas differentiation of multipotential cells was modestly regulated by this variable. (Susan et al., MRS Proceedings, 845, FALL MEETING PROCEEDINGS, AA2.10, 2004). These results may imply that the nanoscale organization of adhesion ligands may be an important variable in controlling cell phenotype and function.

This invention provides a way for a desirable biofunctional peptide to be stoichiometrically incorporated with N-terminal or C-terminal of a mussel adhesive protein, and thus provides more stringent control of density and/or distribution of biofunctional peptide on a substrate while the peptides were randomly incorporated at numerous sites within a target protein in aforementioned methods. The foil length ECM proteins may elicit more significant cellular effects. While short peptide such as CDPGYIGSR (Cys Asp Pro GIy Tyr lie Glys Ser Arg)(SEQ ID NO: 15) has been shown to evoke only 30% of the maximal response obtained by lamininin chemotactic functions with melanoma cells (Gaf et al., Biochemistry, 26, p6896-900, 1987), the use of short polypeptide or oligopeptides creates a more stringent control of substrate conditions such as the surface density or spatial distribution of ligands for cell and/or tissue culturing. Massia & Hubbel disclosed the adhesion of cells onto RGD tri-peptide modified substrates. At very low densities cells attach but do not spread. At higher densities, when the spacing between peptides reaches about 440 nm, cell spread but do not form focal adhesions. However, at still higher densities of RGD peptides, with a spacing of about 140nm or less, spreading is accompanied by development of focal adhesions. This requirement for high density integrin ligands suggests that the interacting integrins need to be clustered in order for focal adhesions to form. (Massia and Hubbell, J Cell Biol, 114, pl089-1100, 1991).

A preferable size range for proteins is from a three amino acids to about 50 amino acids. For any biofunctional peptide, size ranges can be up to about a molecular weight of about 10,000, with a preferable size range being up to a molecular weight of about 5,000, and an even more preferable size range being up to a molecular weight of about 300.

Short peptides are more advantageous because, unlike the long chains that fold randomly upon adsorption causing the active protein domains to be sterically unavailable, short peptides remain stable and do not hide the receptor binding domains when adsorbed. However, the adhesion peptide RGD attached to copolymers having PEO tether lengths of 10 and 22 EO segments showed PEO tether length-dependent cell adhesion activity. Cell spreading and focal adhesion assays revealed that the longer polymer tethers increased the rate of spreading and reduced the time required for fibroblasts to form focal adhesions. (William et al., Biomacromolecules, 8, p3206 -3213, 2007) For this reason, a space linker, but not necessarily may be used to allow the correct formation and/or functioning of the peptide. The space linker may be incorporated between a mussel adhesive protein and a biofunctional peptide. Some specific examples of suitable linkers are given below; it will be evident that the invention is not limited to these particular linkers. Examples are (Gly-Gly-Gly-Gly- Ser)3 (SEQ ID NO: 27) as described in Somia et al., PNAS 90, p7889, 1993, (GIy- Gly-Gly-Gly-Ser)5 (SEQ ID NO: 28), a novel linker, and Asn-Phe-Ile-Arg-Gly-Arg- Glu-Asp-Leu-Leu-Glu-Lys-Ile-Ile-Arg-Gln-Lys-Gly-S er-Ser-Asn (SEQ ID NO: 29) from HSF-I of yeast, see Wiederrecht et al., Cell,54, p841, 1988. Please note in all of embodiments of the present invention, no suitable linker was utilized because the mussel adhesive protein is believed to provide sufficient space for the attached peptide to function.

Alternatively, depending on the usage conditions, the biologically active motif containing peptide fragment can be selected. For example, RGD containing such as Tyr Ala VaI Thr GIy Arg GIy Asp Ser Pro Ala Ser Ser Lys Pro He Ser lie Asn Tyr Arg Thr GIu He Asp Lys Pro Ser GIn Met(SEQ ID NO: 11) which is diclosed in US 4,589,881 can be used.

Cells participating in certain events such as wound healing phenomena are known to release various peptide growth factors. Some of these factors have molecular weight in the range of 10 to 40,000 daltons. For example, activated macrophages secrete numerous growth factors such as NGF and bFGF. Due to the aforementioned problems, peptide mimetic provides more convenient administration and/or conti adhesive nuous feeding. Thus the peptide mimetics recombinantly incorporated into mussel protein provide more convenient administration when compared to an existing ECM mimic that encapsulates cells secreting growth factors or carry these factors.

Most growth factors naturally involved in development and regeneration demonstrate strong binding to the extracellular matrix and are retained there until being locally mobilized by cells (Schmoekel et al., Biotechnology and Bioengineering, 89, p253-262, 2005). For example, tissue regeneration requires both growth factor and extracellular matrix such as collagen, serving as a scaffold for cell growth. A novel protein for controlling cellular functions was constructed by combining functional units of various proteins. The Arg GIy Asp (RGD) sequence functioning as a cell adhesive function, an epidermal growth factor (EGF) as a cell growth function, and a hydrophobic sequence (E 12) as an efficient assembling function, were combined and incorporated into one molecule. (Imen et al., Biomaterials, 27, p3451-3458, 2006).

Thus, growth factors are useful in a number of therapeutic, clinical, research, diagnostic, and drug design applications. However, as previously mentioned, growth factors are typically large. The natural members of the transforming growth factor-β family range upwards of 25 KDa molecular weight. Clinical uses of growth factors, including TGF-βs, may be limited because of their size, such as due to causing immune responses. For example, human TGF-βl is a 25,000 dalton homodimeric protein. In addition to possible adverse immunological responses, large proteins are not often the best candidates for drugs because of the difficulties in administration and delivery.

Consequently, small peptide mimics of natural growth factors which would avoid most of these problems would be desirable for medical applications. It would be advantageous to incorporate peptides mimicking the biological activity of the large, natural members. The invention also provides the ECM mimic that is formed to have the requisite composition to enhance the proliferation and/or differentiation of cells and/or progenitor cells cultured on said ECM mimic. In accordance with one embodiment of the invention, the ECM mimic includes peptide segment derived from naturally occurring growth factors or its peptide mimetics to regulate cell activities. Such peptide segment can also be recombinantly incorporated into a mussel adhesive protein

NGF promotes the survival and activity of certain types of neuronal cells. In addition, NGF promotes the differentiation of premature neuronal cells into postmitotic mature neurons. NGF has been suggested to be effective for treating certain degenerative diseases of both the peripheral and central nervous systems. The administration of NGF may be beneficial in treating diseases in which a deficiency of NGF₅ abnormalities of its receptor, or changes in its transport or intracellular processing lead to a decrease in neuronal function, atrophy or even cell death. Such diseases include hereditary sensory and motor neuropathies, hereditary and sporadically occurring system degeneration, amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease (Goedert et al., MoI. Brain Res., 1, p85— 92, 1986; Mobley et al., Soc. Neurosci. Abstr., 13, pl86, 1987; Mobley et al., Soc. Neurosci. Abstr., 4, p302, 1988; Hefti et al., Ann. Neurol., 20, p275-281, 1986).

However, the bioactivity of NGF, like other proteins, is dependent on its secondary and tertiary structure. The β subunit of NGF has three internal disulfide bonds, which are thought to be important for bioactivity (Kanaya et al., Gene, 83, p65-74, 1989; Iwane et al., Biochem. Biophys. Res. Comm., 171, pll6-122, 1990; Hu and Neet, Gene, 70, p57-65, 1988). In addition, to the extent that any of the protein is denatured, the effective amount of biologically active NGF is diminished. Protein integrity must therefore be maintained during manufacture and storage as well as during administration. The administration of NGF should be sufficient amounts to be therapeutically effective. Another problem in administering NGF as therapeutics should be long lasting for a period of time as aforementioned. These problems associated with its integrity and administration can be overcome when bioactive nerve growth factor mimetic is recombinantly incorporated into a mussel adhesive protein.

NGF interacts selectively with TrkA, BDNF and NT4/5 primarily with TrkB and NT3 mainly with TrkC and, to a lesser extent, also with TrkA and TrkB (Meakin and Shooter, Trends Neurosci., 15, ρ323-331, 1992). NGF mimetic peptides demonstrated good NGF agonist activity at a concentration as low as 3 μM. They induced differentiation of chick dorsal root ganglia and stimulated tyrosine phosphorylation of TrkA, but not TrkB, receptor. (Colangelo et al., J. of Neurosci., 28, 2008).

Therefore, any peptide fragment that selectively binds to TrkA, BDNF, or NT4/5 can be selected to provide an ECM mimic composition according to the present invention. Preferably, the NGF peptide fragment is derived from human natural NGF such as SVSVWVGDKTTATDIKGKEVMVLG (SEQ ID: 24) CTTTHTFVKALTMDGKQAAWRFIR (SEQ ID: 25). More preferably, the peptide is ANVAENA (SEQ ID: 23). Substance P is a neuropeptide: a short-chain polypeptide CRPKPQQFFGLM

(Cys Arg Pro Lys Pro GIn GIn Phe Phe GIy Leu Met)(SEQ ID: 23), that functions as a neurotransmitter and as a neuromodulator. Substance P has been shown to stimulate cellular growth in cell culture (Reid et al., J Cell Biochem, 52, p476-485, 1993), and it was shown that Substance P could promote wound healing of non-healing ulcers in humans (Brown et al., Arch Ophthalmol 115, p926-927, 1997). It has also been shown to reverse diabetes in mice (Dosch et al., Cell, 127, pll23-1135, 2006). This peptide can be recombinantly incorporated into a mussel adhesive for cell and/or tissue culturing.

The invention further provides a method of enhancing wound healing which comprises applying an ECM mimic to a wound.

The presence of growth factor such as NGF may be required for cell or tissue growth. See e.g., Sephel etl al., Biochem Biophys Res Comm, 2, p821-99, 1989. The growth factors may be incorporated into the channel membrane (U.S. Pat. No. 5,011,486), or may be continuously provided within the channel by seeding the channel with cells that secrete the desired growth factors, or a slowly released polymeric gel. (U.S. Pat. Nos. 5,156,844 and 5,106,627). These methods may overcome problems associated with short half lives of various growth factors, and problems with non-continuous or uncontrolled delivery of the factors. In one embodiment, the human umbilical vein endothelial cells (HUVECs) were seeded on a layer of polymerized Matrigel™ (BD Biosciences) with NGF incorporated ECM mimic (10 nM), the endothelial cells, resulting in the capillary tube formation by HUVECs, considering the central role of angiogenesis in development, inflammation, or wound healing. Therefore, a HUVECs seeded ECM mimic would serve as a scaffold for ischemic wound healing. The invention also provides a method of producing an ECM mimic which comprises the steps of: (a) constructing a vector which contains operably a nucleotide sequence encoding a biofunctional polypeptide or oligopeptide;

(b) constructing a transformant by transforming a host cell with the vector; and (c) producing a recombinant adhesive protein by culturing the transformant. E. coli expression system we recently developed (see WO 2005/092920 or WO 2006/107183) can be utilized for scaling-up production of an ECM mimic at economic price, but another expression system can be utilized for scaling-up production.

In one embodiment of the invention, a method of preparing an ECM mimic is provided. E. coli TOPlO [F mcrA Δ(mrr-hsdRMS-mcrBC)Φ801acZΔM15 ΔlacX74 deoR recAl araD139 Δ(ara-leu)7697 galU galK rpsL (St/) endAl nupG] (Invitrogen) may used for recombinant plasmid construction. E. coli BL21(DE3) [F ompT hsdSB (TB mB^~) gal dcm Δ(srl-recA)306: :TnlO(DE3)] (Novagen) was used as a host strain for expressing recombinant proteins. The method comprises the steps of the construction of recombinant plasmid pFP151-ECM derived polypeptide or oligopeptide that can express fusion protein of hybrid fp-151 and a peptide derived from the peptide of which the amino acid sequence is selected from the group consisting of RGD-containing fragment such as GRGDSP, can be constructed. Obviously ECM derived polypeptide or oligopeptide can be from any sources. Preferred fibronectin-, laminin-, collagen-, vitronectin-, heparin binding domain- derived peptide is GRGDSP(SEQ ID NO:2), YIGSR(SEQ ID NO: 11) and/or IKAVA(SEQ ID NO: 16), GVKGDKGNPGWPGAP (SEQ ID NO: 20), GEFYPDLRLKGDK(SEQ ID NO: 21 , and KNNQKSEP(SEQ ID NO: 7), respectively. For construction of production strain and expression of recombinant fp-151-

GRGDSP, E. coli cells were grown in Luria-Bertani (LB) medium. The constructed transformant harboring the recombinant plasmid was stored at -80°C. Cultures were performed in 7 liter LB medium supplemented with 50 μg/ml ampicillin (Sigma) in a 10-liter bioreactor (KoBiotech) at 37⁰C and 250 rpm. Cell growth was monitored by optical density at 600 run (OD₆₀₀) using a ultraviolet (UV)-visible spectrophotometer (UV-1601PC; Shimadzu). When cultures reached an OD₆₀₀ of 0.2-0.5, 1 rnM (final concentration) isopropyl-β-D-thiogalactopyranoside (IPTG) was added to culture broth for induction of fp-151-GRGDSP. The samples were centrifuged at an 18,000 g for 10 min at 4°C and cell pellets were stored at -80°C for further analysis. 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 lysed 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 lysed cell pellet in an acidic organic solvent, preferably an organic solvent with a pH of 3 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 is 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.

Through the method of the invention, usually more than 0.2 g/1 of the ECM mimics of at least 95% purity can be obtained. The solubility of an ECM mimic is significantly high, and thus the ECM mimic is easier to obtain in a concentrated form. For example, an ECM mimic, fp-151-RGD, dissolves in water or 5 % acetic acid solution to a concentration of around 300 mg/ml. The solubility of an adhesive protein is directly related to its ability to stay in highly concentrated forms, so the higher the solubility, the easier it is to make highly concentrated forms with high potential for industrial application.

The ECM mimics obtained through its expression in the invention may 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. To provide stronger adhesion, the tyrosine residues of the extracted fp-151- ECM peptides were converted to DOPA by modification with 50 μg/ml tyrosinase (Sigma) in 0.1 M phosphate-buffered saline (PBS; pH 7) containing 25 mM ascorbic acid and 20 mM sodium borate at 25 °C for 1 h with shaking and aeration (see Taylor, Anal Biochem 302, p70-74, 2002). After modification, the samples were concentrated by ultrafiltration (molecular weight cut off [MWCO] = 10,000 Da, Pharmacia) and dialyzed in 5% acetic acid. To measure the proportion of tyrosine residues modified to DOPA₅ we measured differences in UV spectroscopy (See Waite, Anal Chem, 56, pl935-1939, 1984). The UV absorbance spectrum of DOPA- containing fp-151-RGD can be changed by complexing DOPA with borate at high pH. The absorbencies of 1 mM DOPA standard and modified fp-151-RGD in 0.2 N HCl or 0.2 M sodium borate (pH 8.5) were measured at 292 nm using a UV-visible spectrophotometer. The absorbance differences were measured by subtracting the absorbance of the DOPA standard or modified fp-151-RGD in 0.2 N HCl from that of the modified sample in 0.2 M sodium borate. The 1 mM DOPA standard showed a subtraction difference λ_maχ at 292 nm with a AC value of 3200 M^"1 cm^'1. Using the λ_max and AC of the 1 mM DOPA standard, the number of DOPA residues in the modified fp-151-RGD was calculated according to Beer's law.

The present invention also provides a coating agent which contains the ECM mimic as an active component. Since the ECM mimic of the invention has the characteristic of adhesion, 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 biofouling of the substrates, since the mussel adhesive protein is recombinantly functionalized with cell adhesion inhibitory peptide. An example of application of the coating agent is to coat the motor propeller of ships to prevent biofouling, 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.

The following examples are provided to demonstrate preferred embodiments of the present invention and the invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

EXAMPLE 1 Expression and Purification of fp-151-ECM Peptides

1) Strains and plasmid constructions for fp-151-ECM peptides E. coli TOPlO [F- mcrA Δ(mrr-hsdRMS-mcrBC)Φ801acZΔM15 ΔlacX74 deoR recAl araD139 Δ(ara-leu)7697 galU galK rpsL (Strr) endAl nupG] (Invitrogen) was used for recombinant plasmid construction. E. coli BL21(DE3) [F- ompT hsdSB (rB- mB-) gal dcm Δ(srl-recA)306::Tnl0(DE3)] (Novagen) was used as a host strain for expressing recombinant proteins.

Recombinant plasmid pEDGl 51 -motif that can express fusion protein of hybrid fp-151 and a peptide derived from the peptide of which the amino acid sequence is selected from the ECM protein group, can be constructed. Obviously ECM derived peptide can be from any sources. Preferred fibronectin-, laminin-, collagen-, vitronectin-, heparin binding domain- derived peptide is GRGDSP(SEQ ID NO: 2), YIGSR(SEQ ID NO: 12) and/or IKAVA(SEQ ID NO: 17), GEFYFDLRLKGDK (SEQ ID NO: 20), and KNNQKSEP(SEQ ID NO: 7), respectively. GRGDSP sequence selected from the fibronectin RGD group was added to C-terminus of hybrid fp-151 using polymerase chain reaction (PCR). The fusion protein of hybrid fp-151 and GRGDSP was named as fp-151 -GRGDSP. PCR was performed to generate a 672 bp of fp-151 -GRGDSP encoding fragment using the primers (forward: 5'- GCCATATGGCTAGCGCTAAACCGTCTTAC-3'(SEQ ID NO:33), reverse: 5'-

AAGCTTACGGGCTATCGCCACGGCCTTTGTAAGTCGGGGGG-3'(SEQ ID NO:34)) from pENG151 (Hwang et al., Biomaterials, 114, p3560, 2007) that contains hybrid fp-151 DNA. The amplified fragment was digested with Ndel and Hindlll, and then inserted into the same digested sites of the plasmid vector pET22(b)+ (Novagen) (Fig. 1). The nucleotide sequences of the inserted genes were verified by sequencing.

The other plasmids for the production of other fp-151 -ECM peptides were constructed same way with the case of fp-151 -GRGDSP. The ECM peptides were introduced into C-terminus of fp-151 by PCR using the same forward primer of SEQ ID NO: 33 and backward primers as follows: Table 1: PCR PRIMERS

The amplified fragments were introduced to the parent plasmid pET22(b)+ to produce the plasmids pEDGl 51 -motifs for the other ECM mimics, respectively (Fig. 1). All DNA sequences were confirmed by sequencing.

2) Expression of fp- 151 -ECM peptides

For construction of production strain and expression of recombinant fp-151- ECM peptides, E. colt cells were grown in Luria-Bertani (LB) medium. The constructed cells harboring the recombinant plasmid were stored at -8O⁰C. Cultures were performed in 7 liter LB medium supplemented with 50 μg/ml ampicillin (Sigma) in a 10-liter bioreactor (KoBiotech) at 37°C and 250 rpm. Cell growth was monitored by optical density at 600 nm (OD600) using a ultraviolet (UV)-visible spectrophotometer (UV-1601PC; Shimadzu). When cultures reached an OD600 of 0.2-0.5, 1 mM (final concentration) isopropyl-β-D-thiogalactopyranoside (IPTG) was added to culture broth for induction of fp-151-ECM peptides. The samples were centrifuged at an 18,000 g for 10 min at 4°C and cell pellets were stored at -8O⁰C for further analysis.

3) Purification of fp-151-ECM peptides

Harvested cell pellets were resuspended in 5 ml lysis buffer (10 mM Tris-Cl, 100 mM sodium phosphate, pH 8.0) per gram wet weight. Samples were lysed by constant cell disruption systems (Constant Systems) at 20 KPSI and lysates were centrifuged at 18,000 g for 20 min at 4⁰C and the cell debris was collected for purification. The cell lysate pellet was resuspended in 25% (v/v) acetic acid to extract fp-151-ECM peptides. The extracting solution was centrifuged at 18,000 g for 20 min at 4°C and supernatant was collected and dialyzed in 5% (v/v) acetic acid buffer overnight at 4°C using Spectra/Por molecular porous membrane tubing (MWCO = 10,000 Da, Spectrum Laboratories). Then, the purified sample was freeze dried and stored at -80°C for further analysis.

Samples were resuspended in protein sample buffer (0.5 M Tris-HCl [pH 6.8], 10% glycerol, 5% sodium dodecyl sulfate (SDS), 5% β-mercaptoethanol, 0.25% bromophenol blue) and heated to 100°C for 5 min. After centrifugation for 1 min, proteins were separated by 12% (w/v) SDS-polyacrylamide gel electrophoresis (PAGE) and the detected using Coomassie blue staining (Bio-Rad). Total protein concentration was determined using the Bradford assay (Bio-Rad) with bovine serum albumin (BSA) as a protein standard.

4) MALDI-TOF MS analysis

Matrix-assisted laser desorption ionization mass spectrometry with time-of- flight (MALDI-TOF MS) analysis was performed on a 4700 Proteomics Analyzer (Applied Biosystems) in the positive ion linear mode. Sinapinic acid in 30% acetonitrile and 0.1% trifluoroacetic acid was used as the matrix solution. Samples were diluted 1:25 with matrix solution, and 1 μl of the mixture was spotted onto the MALDI sample target plates and evaporated using a vacuum pump. Spectra were obtained in the mass range between 20,000 and 40,000 Da with -1500 laser shots. Internal calibration was performed using BSA with [M+2H]2+ at 33216. fp-151 -GRGDSP was overexpressed (-25% of total cellular protein) in E. coli in the form of insoluble inclusion bodies (Fig. 2A, lane IS) and was easily purified to -95% purity (Coomassie-blue-stained SDS-PAGE gel analysis) by one- step extraction using 25% (v/v) acetic acid (Fig. 2A, lane AE). The apparent molecular weight of fp-151-GRGDSP on a SDS-PAGE gel was greater than the predicted molecular mass (-28 kDa compared with 25.2 kDa) due to the high pi value (9.89) of fp-151-GRGDSP. We confirmed its correct molecular weight using MADLI-TOF MS analysis of the purified protein sample (Fig. 2B). Other fp-151- ECM peptides including fp-151-laminin(IKVAV), fp-151-laminin(YIGSR), fp-151- collagen, fp-151-substanceP, fp-151 -NGF were also successfully expressed and purified by one-step extraction using 25% (v/v) acetic acid (Fig. 2C). Comparison with fp-151 showed fp-151-ECM peptides to have a similar production yield and simple acetic acid extraction, indicating that fusion of the ECM peptide sequence did not change the superior characteristics of hybrid fp-151 system.

EXAMPLE 2 Cell Adhesion and Spreading Ability of fp-151-ECM Peptides

1) Surface coating

Untreated polystyrene 24-well culture plates (SPL Life Science) were coated with fp-151, fp-151-GRGDSP, fp-151-IKVAV, fp-151 -YIGSR and fp-151 -collagen IV. Cell-Tak (BD Bioscience) and PLL (Sigma) were used as positive controls and uncoated wells were used as negative controls. The amount of coating material used herein was 3.5 μg per cm² of well area. Cell-Tak- and PLL-coated wells were prepared according to the manufacturer's instructions. For fp-151, fp-151-GRGDSP, fp-151-IKVAV, fp-151-YIGSR and fp-151 -collagen IV coated wells were prepared based on the Cell-Tak manufacturer's instruction.

2) Cell adhesion and spreading

Wild-type human HeLa (#CCL-2, ATCC), human kidney epithelial 293T (#CRL-11268, ATCC), Chinese hamster ovary (CHO) (#CCI-61; ATCC), and mouse fibroblast NIH/3T3 (#CRL-1658, ATCC) cells were cultured in Dulbecco's modified Eagle's media (DMEM; Hyclone) supplemented with 10% (v/v) fetus bovine serum (FBS; Hyclone) and penicillin/streptavidin (Invitrogen) at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. All cells for experiments were collected by trypsinization, washed twice in PBS, and diluted to a concentration of approximately IxIO⁵ cells per 1 ml of DMEM without FBS. A total of 5x10⁴ cells (more than 95% of which were viable) in serum-free medium was placed in each 7.5-μg-sample- coated well to see if the coated surface provides biocompatible environment for cell adhesion and spreading.

The cells were allowed to adhere to the coated well surfaces for 1 h and unattached cells were removed from the coated surface by rinsing with PBS. For the cell spreading assay, cells were starved in the serum-deprived medium for 24 h before cell seeding, and incubated on the coated well surfaces for 15 h. In order to visualize the spreading state of cells, cells were fixed with 4% formaldehyde in PBS for 30 min and permeabilized with 0.5% Triton X-100 in PBS for 30 min. After that, actins were labeled with fluorescein isothiocyanate (FITC)-conjugated phalloidin (Sigma) and analyzed using fluorescence microscopy (Olympus).

For quantitative cell-binding measurement after cell adhesion, the 3 -(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed. After coating of each well surface, cell seeding for 1 h, and aspirating medium with unattached cells, 24-well plates were washed with PBS. The MTT assay was performed after 1 h incubation with serum-free DMEM. A total of 300 μl of MTT (Sigma) was added to cover the wells and formazan crystals were allowed to be formed for 2 h. After dissolving formazan crystals with dimethyl sulfoxide (DMSO), the absorbance was measured at 570nm using a microplate reader (Wallac 1420 Victor 3; Perkin Elmer). Each MTT assay was performed in triplicate. Generally, cell attachment and spreading can occur in serum-containing environment regardless of surface coating because many cell attachment and spreading factors, such as fibronectin, vitronectin, and cytokines, are found in serum (Anne et al., J Immunol Methods, 247, p217, 2001). Thus, all cell experiments, except for the cell-proliferation assay, were performed under serum-free conditions. To determine coating topographies, the coated surfaces were investigated by

AFM in tapping mode (Fig. 3). Coating with MAPs significantly changed the surface topographies (Fig. 3B-3E) compared with uncoated surfaces (Fig. 3A). Surfaces coated with modified fp-151 or fp-151-GRGDSP showed similar surface patterns and formed porous structures (Fig. 3C & 3E), whereas unmodified MAPs showed different surface patterns and did not form porous structures (Fig.. 3B & 3D). The porous structures were also observed on the Cell-Tak-coated surface and on the adhesion plaque of mussels where adhesion occurs (Hansen et al., Langmuir, 14, pi 139-1147, 1998), and the proteins in the adhesion plaque contain high levels of DOPA (up to 30% of total amino acids). Therefore, the porous structure might be formed by cross-linking of DOPA residues in the modified proteins. The cell-adhesion ability of fp-151-GRGDSP was also greater than that of fp-151 and all other cell-adhesion materials including Cell-Tak in any type of cell (Fig. 4). Consistent with a previous study, hybrid fp-151 showed greater adhesion ability than PLL but slightly lower adhesion ability than Cell-Tak. Fusion of GRGDSP to the C-terminus of fp-151 improved the cell adhesion ability of fp-151- GRGDSP, such that it is better than Cell-Tak. We surmise that RGD-mediated cell- adhesion ability improved the overall cell-adhesion ability of fp-151 owing to improved cell spreading and formation of an actin cytoskeleton. Other fp-151 -ECM peptide fusion, fp-151 -IKVAV, fp-151 -YIGSR and fp-151 -collagen also showed greater cell-adhesion ability than uncoated condition, but comparable effect to fp-151 , fp-151-GRGDSP, Cell-Tak, and PLL onNIH/3T3 and human 293T cell.

When analyzing the NIH/3T3 cell spreading on the coated surfaces, fp-151- IKVAV, fp-151 -YIGSR, and fp-151 -collagen showed a slightly greater spreading ability than uncoated, fp-151, Cell-Tak, and PLL, but fp-151-GRGDSP showed the greatest spreading ability (Fig. 5). Importantly, fp-151-GRGDSP showed superior cell spreading and attached morphology for all type of cells (additional 293T, CHO and HeLa cells; data not shown) compared with all other cell-adhesion materials assayed. Cell-Tak is a protein mixture extracted from mussel feet where some ECM protein collagens are found, so the Cell-Tak mixture might contain mussel collagens that could improve cell spreading and adhesion (Coyne et al., Science, 277, pi 830, 1997; Qin et al., J Biol Chem, 272, p32623, 1997). By contrast, fp-151 is a pure recombinant protein and does not contain ECM proteins. Interestingly, fp-151- GRGDSP showed good cell spreading in human 293T cells where even Cell-Tak had poor results. In addition, cytoskeleton formation was clearly observed by actin labeling using FITC-conjugated phalloidin in NIH/3T3 cells on a fp-151 -GRGDSP- coated surface (Fig. 5F) but not in cells on surfaces coated with other materials. This demonstrated that fp-151-GRGDSP induced the formation of an actin cytoskeleton and that this improves the ability of cells to make focal adhesions.

EXAMPLE 3 Cell Proliferation Ability of fp-151-ECM Peptides Cell proliferation was evaluated using the MTT assay. A total of 5^χlO⁴ cells (more than 95% of which were viable) of HeLa and CHO cells, 3.75^χl O⁴ cells (more than 95% of which were viable) of 293T cells, or 1.875 x 10⁴ of NIH/3T3 cells were plated onto each 7.5-μg-sample-coated well with DMEM supplemented with 10% FBS and incubated at 37°C for 72 h. Every 24 h, the media were aspirated and 300 μl of MTT was added to the wells to allow formation of formazan crystal for 2 h. Finally, the absorbance was measured at 570 nm using a microplate reader.

The level of cell proliferation on the fp-151-GRGDSP-coated surface was better than for either of the other widely used cell-adhesion materials PLL and CeIl- Tak for tested all cell types (mouse fibroblast NIH/3T3 and human kidney epithelial 293T cell) (Fig. 6), demonstrating that RGD-peptide fusion improves the cell- proliferation ability of fp-151. The levels of cell proliferation on fp-151 -IKVAV-, fp- 151-YIGSR-, and fp-151 -collagen-coated surfaces were also much higher than those on uncoated surfaces, but showed a similar pattern with Cell-Tak-, PLL-, fp-151- and fp-151-GRGDSP-coated surfaces (Fig. 6). In the case of osteoblast MC3T3-E1 cells, all fp-151 -ECM peptides-coated surfaces also showed higher proliferation effects than uncoated and PLL-coated surfaces and had comparable proliferation effects to Cell-Tak-coated surface (Fig. 7).

EXAMPLE 4 Biological Activity Assay of fp-151-NGF

The wounding migration and tube formation activity of the human umbilical vein endothelial cells (HUVECs) were measured as previously described (Lee et al., J Immunol, 177, p5585-5594, 2006). In brief, HUVECs plated at confluence on 60- mm culture dishes were wounded with pipette tips, and then treated with fp-151-NGF (10 nM) and CTGF (connective tissue growth factor; 10 nM) in M 199 medium, supplemented with 2% serum and 1 mM thymidine. After 8 h of incubation, migration was quantified by counting the cells that moved beyond the reference line and photographed (magnification, X50). For the tube formation assay, the HUVECs were seeded on a layer of previously polymerized Matrigel (BD Biosciences) with fp-151-NGF (10 nM) and LK (naturally occurring angiogenic factor; 10 nM). After 18 h of incubation, the cell morphology was visualized via phase-contrast microscopy and photographed (magnification, X40). To measure the formation of the capillary network, the tube length of complete tube networks per field was measured at X40 magnification. The results were expressed as mean ± SD tube length of duplicate wells.

When comparing with non-treated case as a negative control and CTGF- treated case as a positive control, we found that fp-151-NGF exhibited comparable migration activity on HUVECs (Fig. 8). In addition, HUVECs formed the capillary tube by fp-151-NGF treatment compared to non-treatment as a negative control and LK as a positive control (Fig. 9). These data demonstrate that fp-151-NFG ECM mimics might play a central role of angiogenesis in development, inflammation, or wound healing.

Claims

WHAT IS CLAIMED IS:

1. A chimeric polypeptide comprising a mussel adhesive protein and a biofunctional peptide coupled to the mussel adhesive protein.

2 The chimeric polypeptide of claim 1, wherein the biofunctional peptide is linked to C-terminus, N-terminus or C- and N-terminus of the mussel adhesive protein.

3. The chimeric polypeptide of claim 1, wherein the mussel adhesive protein is a fusion protein comprising a first peptide of mussel foot protein (FP)-5 and a second peptide of at least one selected from the group consisting of mussel FP-I, mussel FP-

3. mussel FP-4 and fragment thereof, and the second peptide is linked to C-terminus, N-terminus or C- and N-terminus of the FP-5.

4. The chimeric polypeptide of claim 3, wherein the FP-5 comprises SEQ ID NO: 30.

5. The chimeric polypeptide of claim 3, wherein the mussel adhesive protein comprises FP-5 and FP-I, or FP-5 and FP-3.

6. The chimeric polypeptide of claim 5, wherein the FP-I comprises an amino acid sequence of SEQ ID NO: 32 tandemly repeated 1 to 80 times.

7. The chimeric polypeptide of claim 3, wherein the FP-3 comprises SEQ ID NO: 31.

8. The chimeric polypeptide of claim 1, wherein the biofunctional peptide is derived from extracellular matrix protein.

9. The chimeric polypeptide of claim 8, wherein the extracellular matrix protein is fibronectin, laminin, collagen, vitronectin, substance P, or nerve growth factor.

10. The chimeric polypeptide of claim 9, wherein the biofunctional peptide is derived from a cell binding domain or heparin binding domain of fibronectin.

11. The chimeric polypeptide of claim 10, wherein the biofunctional peptide is selected from the group consisting of peptides comprising an amino acid sequence of SEQ ID NOs:l to ll.

12. The chimeric polypeptide of claim 11, wherein the biofunctional peptide is a peptide having an amino acid sequence of SEQ ID NO: 2.

13. The chimeric polypeptide of claim 9, wherein the biofunctional peptide is derived from laminin.

14. The chimeric polypeptide of claim 13, wherein the biofunctional peptide is selected from the group consisting of peptides comprising an amino acid sequence of SEQ ID NOs: 12 to 18.

15. The chimeric polypeptide of claim 14, wherein the laminin-derived peptide is a peptide having an amino acid sequence of SEQ ID NO: 12 or a peptide having an amino acid sequence of SEQ ID NO: 17.

16. The chimeric polypeptide of claim 9, wherein the biofunctional peptide is derived from collagen type I or collagen type IV.

17. The chimeric polypeptide of claim 16, wherein the biofunctional peptide is a collagen-derived peptide selected from the group consisting of peptides comprising an amino acid sequence of SEQ ID NOs: 19 to 21.

18. The chimeric polypeptide of claim 9, wherein the biofunctional peptide is a vitonectin-derived peptide comprising an amino acid sequence of SEQ ID NO:22, a substance P-derived peptide comprising an amino acid sequence of SEQ ID NO:23, or a nerve growth factor-derived peptide selected from the group consisting of peptides comprising an amino acid sequence of SEQ ID NOs:24 to 26.

19. A bioadhesive extracellular matrix comprising the chimeric polypeptide which comprises a mussel adhesive protein comprising and a peptide derived from an extracelluar matrix protein coupled to the mussel adhesive protein.

20 The bioadhesive extracellular matrix of claim 19, wherein the spatial distribution or surface density of extracellular matrix protein is controlled on a substrate.

21 The bioadhesive extracellular matrix of claim 19, wherein the peptide derived from an extracelluar matrix protein is linked to C-terminus, N-terminus or C- and N-terminus of the mussel adhesive protein.

22. The bioadhesive extracellular matrix of claim 19, wherein further comprises cells.

23. The bioadhesive extracellular matrix of claim 19, wherein the mussel adhesive protein is a fusion protein comprising a first peptide of mussel foot protein

(FP)-5 and a second peptide of at least one selected from the group consisting of mussel FP-I, mussel FP -3, mussel FP-4 and fragment thereof, and the second peptide is linked to C-terminus, N-terminus or C- and N-terminus of the FP-5.

24. The bioadhesive extracelluar matrix of claim 23, wherein the FP-5 comprises SEQ ID NO: 30.

25. The bioadhesive extracelluar matrix of claim 23, wherein the mussel adhesive protein comprises a fusion protein of FP-5 and FP-I, or a fusion protein of FP-5 and FP-3.

26. The bioadhesive extracelluar matrix of claim 23, wherein the FP-I comprises an amino acid sequence of SEQ ID NO: 32 tandemly repeated 1 to 80 times.

27. The bioadhesive extracelluar matrix of claim 23, wherein the FP-3 comprises SEQ ID NO: 31.

28. The bioadhesive extracelluar matrix of claim 21, wherein the extracellular matrix protein is fibronectin, laminin, collagen, vitronectin, substance P, or nerve growth factor.

29. The bioadhesive extracelluar matrix of claim 28, wherein the peptide is derived from a cell binding domain or heparin binding domain of fibronectin.

30. The bioadhesive extracelluar matrix of claim 29, wherein the peptide is selected from the group consisting of peptides comprising an amino acid sequence of SEQ ID NOs:l to ll.

31. The bioadhesive extracelluar matrix of claim 30, wherein the peptide is a peptide having an amino acid sequence of SEQ ID NO: 2.

32. The bioadhesive extracelluar matrix of claim 31, wherein the peptide is derived from laminin.

33. The bioadhesive extracelluar matrix of claim 32, wherein the laminin- derived peptide is selected from the group consisting of peptides comprising an amino acid sequence of SEQ ID NOs: 12 to 18.

34. The bioadhesive extracelluar matrix of claim 33, wherein the peptide has an amino acid sequence of SEQ ID NO: 12 or an amino acid sequence of SEQ ID NO:

17.

35. The bioadhesive extracelluar matrix of claim 28, wherein the peptide is derived from collagen type I or collagen type IV.

36. The bioadhesive extracelluar matrix of claim 35, wherein the peptide is a collagen-derived peptide is selected from the group consisting of peptides comprising an amino acid sequence of SEQ ID NOs: 19 to 21.

37. The bioadhesive extracelluar matrix of claim 28, wherein the biofunctional peptide is a vitonectin-derived peptide comprising an amino acid sequence of SEQ ID NO:22, a substance P-derived peptide comprising an amino acid sequence of SEQ ID NO:23, or a nerve growth factor-derived peptide selected from the group consisting of peptides comprising an amino acid sequence of SEQ ID NOs:24 to 26.

38. A coating substrate comprising: a substrate; and a coating formed on the substrate by a coating solution comprising a mixture of a chimeric polypeptide according to any one of claims 1 to 18, and an aqueous solvent.

39. The coating substrate of Claim 38, wherein the density and/or distribution of biofunctional peptide on the substrate.

40. A method of controlling an adhesiveness of extracellular matrix protein which comprises a step of preparing a chimeric polypeptide of a mussel adhesive protein and a peptide derived from an extracellular matrix protein coupled to the mussel adhesive protein.