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Smart bioadhesive materials from marine mussel
What man-made good exists that provides us both life’s necessity and luxury? A popular seafood cuisine, mussels are enjoyed all around worlds. They can be smoked, boiled, or steamed to be served alone or as an ingredient of a large dish. It can be also produce beautiful pearls and jewelry. Now scientists have discovered another use for nature’s gift.

Our MAGIC team has developed a novel type of practical recombinant hybrid bioadhesive material that is originated from marine mussel.

Mussels produce and secrete specialized adhesives that work in water allowing them to attach themselves in rough marine environments. These mussel adhesive proteins (MAPs) have been studied as a potential source of water-resistant bioadhesives for the past 25 years. They adhere tightly to substrata using the byssus, which is secreted from their foot and comprises a bundle of threads. At the end of each thread, there is an adhesion plaque containing a water-resistant adhesive that enables the plaque to anchor to wet, solid surface. Strong and water-insoluble mussel adhesives have attracted interest for potential uses in biotechnological applications because they could be used as cell, tissue, or medical adhesives and have the added advantage of being environmentally friendly. This adhesion plaque is composed of five distinct types of protein - foot proteins type 1 (fp-1) to type 5 (fp-5).

At present, Cell-Tak, which is a mixture of extracted MAPs and comprises mainly fp-1 and fp-2 is the only commercially available MAPs. However, the methods by which these MAPs are produced are inefficient and uneconomical: about 10,000 mussels are required to obtain one gram of Cell-Tak. As a result, high production costs can limit use, and they are only used as cell and tissue-culture adhesion agents. Therefore, recombinant DNA technology has been used and mass production of MAPs has been attempted in several expression systems. However, attempts to produce a functional MAP have failed for several reasons. Even though recombinant fp-1 decapeptide repeats have been successfully expressed as insoluble inclusion bodies in E. coli and extracted using acetic acid solution, their adhesion properties have not been fully addressed. From the mid-1990s, attempts have also been made to produce synthetic polypeptide mimics of fp-1, but these mimics have not shown biocompatibility data.

fp-5 and fp-3, which are located at the interface between the substratum and the adhesion plaque of mussels, have been discovered during the past 10 years and they have been found to contain high levels of DOPA (L-3,4-dihydroxyphenyl alanine); indeed, the DOPA content is linearly correlated with the adhesion strength of MAPs. Previously, we have successfully shown that recombinant fp-5 with functional adhesion properties can be produced from Escherichia coli. Especially, recombinant fp-5 showed superior adhesion ability to Cell-Tak. However, soluble expression of recombinant fp-5 inhibited cell growth and led to a low production yield. Purification of fp-5 was also found to be complicated due to its adhesive property and the purification yield was very low. In addition, recombinant fp-5 was highly insoluble in aqueous buffer after purification, and thus, preparation of the highly concentrated solution required for practical use was not possible. In this research, to overcome several of the limitations of previous MAPs, Professor Cha and his team designed and produced the novel type of hybrid MAP fp-151, which is a fusion protein comprising six fp-1 decapeptide repeats added to the N- and C-termini of fp-5 in E. coli cells. Using micro- and bulk-scale characterization and mammalian/human cell-adhesion analyses, they demonstrated that hybrid fp-151 has the potential to be a practical bioadhesive with strong adhesive ability (~10 kg subject can be attached on 1 cm2 area using 40 mg hybrid fp-151), a simple purification process (~1 g-purified protein per 1 liter-pilot-scale fed-batch bioreactor culture), proper manipulation properties (~300 g/l solubility), and high biocompatibility.

This novel hybrid mussel bioadehsive materials are now in commercialization stage as cell and tissue bioadhesive (first item) under collaboration with Kollodis Biosciences.
Development of bioadhesives from marine mussels
Mussel adhesive proteins have received increased attention as potential biomedical and environmentally friendly underwater adhesives thanks to their fascinated properties, including strong and flexible adhesion, adhesion to various material substrates, water displacing, harmless to human body, and controlled biodegradability. Several mussel adhesive proteins have been identified and characterized from mussels, and profound biochemical knowledge for mussel adhesions has been accumulated. In addition, lots of efforts have been attempted to realize these promising bioadhesive materials from marine mussels. Here, progresses in diverse developmental approaches, with particular emphasis on functional production of mussel adhesive proteins, are being reviewed.

1.Introduction
Many marine ‘fouling’ organisms, especially marine invertebrates, live in dynamic ocean environment by adhering tightly to underwater substratum using their holdfasts. Among these sessile marine organisms, mussels and barnacles have become major target for marine adhesion studies since they are easily seen due to their huge numbers. While studies on barnacle adhesion are still in relatively early stage [1,2], lots of biochemical knowledge for mussel adhesions has been accumulated during the over past 25 years.
   Mussels, which are a common food throughout the world, have been studied as a potential source for a water-resistant bioadhesive [3,4]. Mussels produce and secrete specialized adhesives that function in water allowing them to attach themselves in marine environments, which are characterized by salinity, humidity, tides, turbulence, and waves [3]. They adhere tightly to surfaces underwater using the byssus secreted from their foots, which consist of a bundle of threads (Fig. 1A). 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 [5]. These mussel adhesive proteins have been considered as a potential underwater bioadhesives due to their many fascinated features [3,6-8]. Mussel adhesive proteins are known as the most powerful adhesion in the world. Even though mussel adhesion is much stronger than other polymer-based adhesives such as epoxy and phenolic resins, it is flexible and elastic. Most importantly, the best fascination of mussel adhesive protein is that maintenance of its adhesion in wet environment. All adhesive events in living organisms occur in the presence of water, but adhesions using chemical-based adhesives are easily broken by water. There are no synthetic (chemical-based) adhesives that can be similarly applied an underwater environment. Mussel adhesive protein can also attach to various substances, including plastic, glass, metals, Teflon, and living body substances. Adhesions of mussel adhesive proteins (extracted and recombinant) to various solid materials have already been verified [9-14]. Recent studies on attaching of mussel adhesive proteins to living body substances, especially porcine skin [15], and diverse types of mammalian cells [13,16,17] have been also executed. Strong and water-insoluble mussel adhesives have attracted interest for potential uses in diverse biotechnological applications because they could be used as cell and tissue adhesives, and have the added advantage of being environmentally friendly from biodegradability [18]. Mussel adhesive proteins can also be used as medical adhesives as they are harmless to the human body, and do not impose immunogenicity [6,8,18,19]. Therefore, lots of efforts have been tried to realize practical mussel bioadhesives. In this article, we review progress in diverse developmental approaches, with particular emphasis on production of mussel adhesive proteins and their characteristics.


Figure 1. (A) Attachment of mussel to other mussel using byssal threads.
(B) Schematic illustration of a byssal thread and adhesive plaque.
2. Mussel adhesive proteins
Discovery and characterization of mussel adhesive proteins have been mainly lead by Dr. Waite's group ( University of California , Santa Barbara , USA ). The byssus and the adhesive plaque of mussels are composed of several different proteins. Studies on mussel adhesive proteins and their characteristics have identified three distinct types of collagenous proteins from byssus thread [20-22] and six distinct types of proteins, foot proteins type 1 (fp-1) to type 6 (fp-6), from adhesive plaque (Table 1) [5,9,23-27]. The thread is dominated throughout by collagen-silk hybrids known as preCols [22]. Mytilus edulis has been major target mussel for discovering foot proteins, but it was reported that other mussels such as Mytilus galloprovincialis , Mytilus coruscus , and Mytilus californiaus also have similar types and amino acid sequences of mussel adhesive proteins [10,18,28]. Interestingly, these foot proteins contain high levels of 3,4-dihydroxyphenyl-L-alanine (Dopa), which is a catabolic amino acid that is produced by post-translational hydroxylation of tyrosine using polyphenol oxidase [5,23,24]. 3,4-dihydroxyphenyl (catechol), side chain of Dopa, is able to form strong hydrogen bonds with hydrophilic surfaces and strong complexes with metal ions, metal oxides (Fe 3+ , Mn 3+ ), and semimetals (silicon) [28-30]. Mussel adhesive proteins that are closer to the adhesion interface have a higher proportion of Dopa residues [23-26]. It was also reported that mussel adhesive protein analogs without Dopa showed greatly reduced ability for adhesion [10,11,31]. Dopa residues also enable mussel adhesive protein molecules to cross-link each other by oxidative conversion to Dopa-quinone [11,31]. The reactive quinine is thought to provide the water-resistance characteristics of mussel adhesion [11,31]

Table 1. Mussel adhesive proteins

Proteins Mass (kDa) DOPA (mol%) Features
fp-1 ~110 ~13

~80 times repeats of AKPSYPPTYK
two variants, basic proteinsurface coating

fp-2 ~40 ~3 Cysteine rich
abundant in adhesion plaque
fp-3 ~6 ~20 arginine rich, hydroxyarginine
20-30 variants
surface adhesion
fp-4 ~80 ~5 histidine, lysine, arginine rich
Cu2+ binding
fp-5 ~9.5 ~30 phosphoserine
YK or YH repeats
surface adhesion
fp-6 ~11 ~4 cysteine rich (~11 mol%)
Isolated from M. edulis and M. galloprovincialis except fp-6 that is discovered in M. californiaus

   Foot protein fp-1 from M. edulis (Mefp-1) was firstly identified and is the most studied mussel adhesive protein [25,32]. Mefp-1 has peculiar structure of about 80-times repeats of decapeptide (Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Tyr-Dopa-Lys). Thus, it has high molecular weight of ~110 kDa and its Dopa content is 10-15 mol%. Covering the entire structure of adhesion plaque amd distal portion of thread is a thin protective cuticle comprised largely of Mefp-1 (Fig. 1B) [18]. Other Mytilus mussel species also contain a protein analogous to Mefp-1 with slight differences in decapeptide repeat frequency and amino acid composition; Mgfp-1 from M. galloprovincialis [33], Mcfp-1 from M. coruscus [34], fp-1 from M. californiaus [35].
   The bulk of adhesion plaque in mussel is formed by Mefp-2 and Mefp-4 (Fig. 1B). Mefp-2 has about 40 kDa molecular weight with ~3 mol% Dopa contents and is relatively resistant to a variety of proteolysis that is an important feature for integrity of the byssal plaque. In addition, Mefp-2 has epidermal growth factor-like motifs that might have stabilization role in the byssus [26]. Mefp-4 has ~80 kDa molecular weight with ~4 mol% Dopa contents and high levels of histidine, lysine, and arginine. This protein seems to serve as a coupling agent in the thread plaque junction [36].
   Mefp-3 and Mefp-5, which are located at the interface between the substratum and the adhesion plaque of mussels (Fig. 1B), have been discovered during the past 10 years and they have been found to contain higher levels of Dopa. Mefp-3 contains high Dopa level at ~20 mol% and is basic protein having a large number of arginine residues [23,36]. It is the smallest protein having mass of ~6 kDa but has many (20~30) variants. Currently, only four or five variants have been detected in plaques. Specific variant has been though to be dependent on the surface used for attachment, but it was reported that no correlation between Mefp-3 expression and surface type [37]. Other mussels also contain protein analogous: Mgfp-3A and Mgfp-3B from M. galloprovincialis [34] and Mcfp-3-12 variants from M. californiaus [38]. Mefp-5 is a plaque specific protein and contains the highest Dopa level at ~30 mol% among mussel adhesive proteins [24]. This Mefp-5 also has many serine residues (8 of total 75 amino acids) that can be partly modified to phosphoserines. It is basic protein (a large number of lysine residues) having an isoelectric point (pI) of 8.3 (with phosphorylation) and has mass of ~9.5 kDa. But, the role of phosphoserine in mussel adhesion is not revealed exactly [18]. Recently, cDNA of Mgfp-5 from M. galloprovincialis was also cloned and its nucleotide sequence had ~94% homologous with that of Mefp-5 [10]. More recently, Mcfp-5 from M. californianus with mass of 8.9 kDa, was identified [27].
   New type of mussel adhesive protein Mcfp-6 from M. californianus, with mass of 11.6 kDa, was also identified [27]. Mcfp-6 is basic protein and contains a small amount of Dopa (<5 mol%). In contrast to Mcfp-3 and Mcfp-5, tyrosine prevails at 20 mol%, and cysteine is present at 11 mol%, one-third of which remains thiolate [27]. It is believed that Mcfp-6 may provide a cohesive link between the surface-coupling Dopa-rich proteins and the bulk of the plaque proteins.

3. Production of mussel adhesive proteins
Even though mussel adhesive protein has many fascinating properties and applicable fields, the practical applications of mussel adhesive protein are quite restricted due to its extremely limited production. Although natural extraction was initially used to isolate mussel adhesive proteins for commercial purposes, this process is labor-intensive and inefficient, requiring around 10,000 mussels for 1 g Mefp-1 (Table 2) [39,40]. In addition, the chemical extraction process does not always yield pure or individual adhesive proteins. At present, Cell-Tak™ (BD Bioscience Clontech), which is a extracted mixture and comprises mainly Mefp-1 and Mefp-2, and MAP™ (Swedish BioScience Lab.), which is extracted Mefp-1, are the only commercially available mussel adhesive proteins. Low extraction yield and high production costs can limit use, and they are only used as cell and tissue-culture adhesion agents. Therefore, recombinant DNA technology has been used to obtain large amounts of adhesive protein components for further conventional adhesion tests and practical applications. Genetic production of fp-1 has been attempted in several expression systems, including Escherichia coli, yeast, and plants (Table 2) [9,39,41] because fp-1 has been considered a key protein for adhesion of mussels in wet environments. However, attempts to produce a functional, practical mussel adhesive protein (mainly full size of fp-1) have failed for several reasons, including a highly biased amino-acid composition (five amino-acid types comprise ~89% of the total amino acids; Table 3), different codon usage preference between mussel and other expression systems (tRNA utilization problem), and small expression quantity [12,39,41]. Even though partial fp-1 decapeptide repeats (usually 6~20) have been successfully expressed as insoluble inclusion bodies in Saccharomyces cerevisiae [9] and E. coli [12,41,42] using synthetic gene constructs, their adhesion properties have not been fully addressed. Expression of full size of Mgfp-1 was also tried in transgenic tobacco plant because of its similarity to the repetitive plant cell wall proteins [43], but further reports on production levels and adhesion ability were not followed after that. In addition, intrinsic problems of plant cell culture such as low production levels and considerable expense in cell culture techniques should be solved for industrial production. Culturing of mussel primary cells has also been attempted, but the production yield of mussel adhesive protein was not reported [44]. Thus, we can conclude that successful mass production technologies were not developed for functional recombinant mussel adhesive proteins before our recent successful reports [10,13]. From the mid-1990s, attempts have also been made to produce synthetic polypeptide mimics of fp-1, but these mimics have not shown biocompatibility as natural mussel adhesive proteins [11,31,45,46].

Table 2. Production of mussel adhesive proteins and its mimics

System Feature Ref

Natural extraction

Mefp-1
Easy extraction process
Labor intensive (10,000 mussel/g)
High production cost
25
Yeast Partial fp-1 (20 repeats)
Low production level
No hydroxylation
Low adhesion strength
9
E. coli Partial fp-1 repeats (6~20 repeats)
Inclusion body formation
High production is possible
No hydroxylation
Low adhesion strength
12,41,42
Mgfp-5
Soluble expression
Low production level
Micro-scale adhesion tests
Strong adhesion strength
10
Mgfp-3A
Low production level
Micro-scale adhesion tests
Medium adhesion strength
47
Hybrid fp-151
Inclusion body formation
High production level
Easy extraction process
Macro-scale adhesion tests
Strong adhesion strength
13
Plant

Full fp-1
Low production level

43

Polymer
based mimics

Dopa-containing chemical-based polymer
Functional production
Biocompatibility problem

11,31,45,46

Table 3. Amino acid distribution of fp-1

Amino acid Number Mol%
Lys 190 21.7
Pro 224 25.6
Ser 87 9.9
Thr 107 12.2
Tyr 168 19.2
Others 99 11.4
Total 875 100

     A recent study reported the identification of Mefp-5 [24], and it appears this protein may be used as a potent bioadhesive since it has the highest known Dopa content among the discovered mussel adhesive proteins. However, adhesion characteristics of Mefp-5 protein were not be investigated due to its very limited purified amount. Recently, our laboratory have successfully shown that Mgfp-5 and Mgfp-3A with functional adhesion properties can be produced from recombinant E. coli system [10,47]. Especially, recombinant Mgfp-5 showed superior adhesion ability to Cell-Tak™ and recombinant Mgfp-3A when several micro-scale analytical tools, including coatings on diverse surfaces, quartz crystal microbalance (QCM), atomic force microscopy (AFM), and diverse cell adhesion on culture surface, were used [10,17]. However, soluble expression of recombinant Mgfp-5 inhibited cell growth after induction and led to a low production yield (Table 4). Purification of Mgfp-5 was also found to be complicated due to its adhesive property and thus, the purification yield was very low. In addition, recombinant Mgfp-5 was highly insoluble in aqueous buffer after purification. Therefore, preparation of the highly concentrated solution required for practical use was not possible. Recombinant Mgfp-3A has been shown to have similar adhesion ability as Cell-Tak™, but expression of Mgfp-3A also severely inhibited cell growth and led to a very low production yield.

Table 4. Comparison of several mussel adhesive proteins.

Material Expression Yield a (%) Purification Yield b (%) Production
Yieldc (mg/L)
Solubility c
(g/L
Cell Adhesion Cell Spreadingd Ref
Mgfp-1 N/A e N/A N/A N/A Excellent (Cell-Tak) Medium (Cell-Tak)  
Mgfp-5 ~13 ~7 ~2 ~1 Good Poor 10,17
fp-151 ~40 ~53 ~1000 ~300 Excellent Poor 13
fp-151-RGD ~40 ~53 ~1000 ~300 Excellent Excellent 16
aBased on total cellular proteins
bBased on initial mussel adhesive protein
cAfter purification
dDeformation of the plasma membrane and formation of cell?substrate attachments
eNot applicable

   Therefore, to overcome several of the limitations of these proteins, the novel hybrid type of mussel adhesive protein fp-151, which is a fusion protein comprising six Mgfp-1 decapeptide repeats added to the N- and C-termini of Mgfp-5, was designed and produced in E. coli [13]. This hybrid mussel bioadhesive fp-151 showed significant greater production yields and easier purification using acetate extraction (Figure 2 & Table 4). Pilot-scale (200 liter) production also showed the possibility of economical mass production of fp-151 (~1 g-purified protein per 1 liter-fed-batch bioreactor culture). Purified recombinant fp-151 had comparable adhesion characteristics to recombinant Mgfp-5 through micro-scale adhesion analyses including surface coating, QCM, and AFM. Adhesion to laboratory plastic consumables was also tested, and hydroxylated fp-151 using tyrosinase reaction easily adhered to these items within 10 min but it took about 12 h for complete cross-linking. This new fp-151 protein also showed efficient adhesion (Figure 3) and good biocompatibility for various cell types including both anchorage-independent and anchorage-dependent cells [13]. Moreover, because the post-purification insolubility problems were overcome with fp-151, sufficient concentrations (~300 g/L) of adhesive solution were obtained (Table 4). Therefore, macro-scale adhesion strengths of recombinant fp-151 was possible and a comparative study with the commercially available tissue adhesive fibrin glue as a positive control showed that the shear strength of fp-151 was about 4-times greater (~0.8 MPa) than that of fibrin glue (~0.2 MPa) when cowhide square pillars (10 mm x 10 mm) and 10 mg samples were used (Figure 4).


Figure 2. (A) Expression and purification of hybrid fp-151 in recombinant E. coli. Coomassie-blue-stained SDS-PAGE analysis was performed. Lanes: MW, protein molecular weight marker; WC, whole-cell sample; IS, insoluble cell debris fraction; AE, fraction extracted with 25% (v/v) acetic acid; AF, eluted fraction using His-tag affinity chromatography. (B) Purified and lyophilized recombinant hybrid fp-151 (Kollodis Bioscience).


Figure 4. (A) Schematic of equipment used for the adhesion strength test, with cowhide adherends, in the Instron force measurement system with a 500 N (maximum capacity) load cell. (B) Comparison of the shear strength of hybrid fp-151 and fibrin glue in cowhide square pillars. The total amount of protein applied was 10 mg per cowhide square pillar (10 mm x 10 mm). The fixtures were cured at 45°C for 6 h in air. Each value and error bar represents the mean of four independent experiments and the standard deviation.


Figure 5. Cell spreading of human HeLa, human 293T, and Chinese hamster ovary (CHO) cells on (A) uncoated (NC), (B) PLL-, (C) Cell-Tak TM -, (D) fp-151-, and (E) fp-151-RGD-coated polystyrene surfaces. Bare polystyrene surfaces were coated with 7.5 μg of each sample and 5×10 4 cells (more than 95% of which were viable) in serum-free medium added to each coated well and incubated for 18 h. The scale bar is 100 m m.

   Our laboratory has also developed other hybrid type of mussel adhesive protein for specific application as cell-adhesion biomaterial [16]. We designed and constructed fp-151-RGD by fusing the GRGDSP peptide, one of the RGD peptides at the cell attachment site of fibronectin, with the C-terminus of fp-151 to utilize cationic and DOPA-mediated forces from mussel adhesive proteins and integrin-mediated force from the RGD peptide. New hybrid fp-151-RGD had the advantages of fp-151, such as high production yield and simple purification, but also showed superior cell-adhesion and spreading abilities under serum-free conditions regardless of mammalian cell type compared with other widely used cell-adhesion materials such as poly-L-lysine (PLL) and Cell-Tak™ (Figure 5 & Table 4). Collectively, these novel hybrid type mussel adhesive proteins, fp-151 and fp-151-RGD, have the potential to become practical bioadhesives for medical, bioscience, and biotechnological applications.

4. Concluding remarks
Mussel adhesive proteins have potential as environmentally friendly adhesives for use under aqueous conditions and may be of particular value in medical applications. During the last two decades, many efforts have been tried to develop bioadhesives from marine mussels. However, practical applications of Dopa-containing mussel adhesive proteins have been severely limited by uneconomical extraction and unsuccessful large-scale production. Availability of large quantities of recombinant mussel adhesive proteins will enable to develop practical bioadhesives for diverse applications. Recent developed new hybrid types of mussel-inspired adhesive proteins might enable to realize this dream. Therefore, the researchers need to use several adhesive proteins simultaneously to develop bioadhesive materials with more practical and better properties, and based on these developed bioadhesives, they should find novel biological applications including gene and drug delivery, anti-biofouling coatings, medical device coatings, and surgical sealants.

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