Dong Soo Hwang

Strong reversible Fe3+-mediated bridging between dopa-containing protein films in water

Metal-containing polymer networks are widespread in biology, particularly for load-bearing exoskeletal biomaterials. Mytilus byssal cuticle is an especially interesting case containing moderate levels of Fe3+ and cuticle protein—mussel foot protein-1 (mfp-1), which has a peculiar combination of high hardness and high extensibility. Mfp-1, containing 13 mol % of dopa (3, 4-dihydroxyphenylalanine) side-chains, is highly positively charged polyelectrolyte (pI ∼ 10) and didn’t show any cohesive tendencies in previous surface forces apparatus (SFA) studies. Here, we show that Fe3+ ions can mediate unusually strong interactions between the positively charged proteins. Using an SFA, Fe3+ was observed to impart robust bridging (Wad ≈ 4.3 mJ/m2) between two noninteracting mfp-1 films in aqueous buffer approaching the ionic strength of seawater. The Fe3+ bridging between the mfp-1-coated surfaces is fully reversible in water, increasing with contact time and iron concentration up to 10 μM; at 100 μM, Fe3+ bridging adhesion is abolished. Bridging is apparently due to the formation of multivalent dopa-iron complexes. Similar Fe-mediated bridging (Wad ≈ 5.7 mJ/m2) by a smaller recombinant dopa-containing analogue indicates that bridging is largely independent of molecular weight and posttranslational modifications other than dopa. The results suggest that dopa-metal interactions may provide an energetic new paradigm for engineering strong, self-healing interactions between polymers under water.

Protein- and Metal-dependent Interactions of a Prominent Protein in Mussel Adhesive Plaques

The adhesive plaques of Mytilus byssus are investigated increasingly to determine the molecular requirements for wet adhesion. Mfp-2 is the most abundant protein in the plaques, but little is known about its function. Analysis of Mfp-2 films using the surface forces apparatus detected no interaction between films or between a film and bare mica; however, addition of Ca2+ and Fe3+ induced significant reversible bridging (work of adhesion Wad ≈ 0.3 mJ/m2 to 2.2 mJ/m2) between two films at 0.35 m salinity. The strongest observed Fe3+-mediated bridging approaches the adhesion of oriented avidin-biotin complexes. Raman microscopy of plaque sections supports the co-localization of Mfp-2 and iron, which interact by forming bis- or tris-DOPA-iron complexes. Mfp-2 adhered strongly to Mfp-5, a DOPA-rich interfacial adhesive protein, but not to another interfacial protein, Mfp-3, which may in fact displace Mfp-2 from mica. In the presence of metal ions or Mfp-5, Mfp-2 adhesion was fully reversible. These results suggest that plaque cohesiveness depends on Mfp-2 complexation of metal ions, particularly Fe3+ and also by Mfp-2 interaction with Mfp-5 at the plaque-substratum interface.

Expression of Functional Recombinant Mussel Adhesive Protein Mgfp-5 in Escherichia coli

ABSTRACT Mussel adhesive proteins have been suggested as a basis for environmentally friendly adhesives for use in aqueous conditions and in medicine. However, attempts to produce functional and economical recombinant mussel adhesive proteins (mainly foot protein type 1) in several systems have failed. Here, the cDNA coding for Mytilus galloprovincialis foot protein type 5 (Mgfp-5) was isolated for the first time. Using this cDNA, we produced a recombinant Mgfp-5 fused with a hexahistidine affinity ligand, which was expressed in a soluble form in Escherichia coli and was highly purified using affinity chromatography. The adhesive properties of purified recombinant Mgfp-5 were compared with the commercial extracted mussel adhesive Cell-Tak by investigating adhesion force using atomic force microscopy, material surface coating, and quartz crystal microbalance. Even though further macroscale assays are needed, these microscale assays showed that recombinant Mgfp-5 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments.

Adhesion of mussel foot proteins to different substrate surfaces.

Mussel foot proteins (mfps) have been investigated as a source of inspiration for the design of underwater coatings and adhesives. Recent analysis of various mfps by a surface forces apparatus (SFA) revealed that mfp-1 functions as a coating, whereas mfp-3 and mfp-5 resemble adhesive primers on mica surfaces. To further refine and elaborate the surface properties of mfps, the force–distance profiles of the interactions between thin mfp (i.e. mfp-1, mfp-3 or mfp-5) films and four different surface chemistries, namely mica, silicon dioxide, polymethylmethacrylate and polystyrene, were measured by an SFA. The results indicate that the adhesion was exquisitely dependent on the mfp tested, the substrate surface chemistry and the contact time. Such studies are essential for understanding the adhesive versatility of mfps and related/similar adhesion proteins, and for translating this versatility into a new generation of coatings and (including in vivo) adhesive materials.

Viscosity and interfacial properties in a mussel-inspired adhesive coacervate.

The chemistry of mussel adhesion has commanded the focus of much recent research activity on wet adhesion. By comparison, the equally critical adhesive processing by marine organisms has been little examined. Using a mussel-inspired coacervate formed by mixing a recombinant mussel adhesive protein (fp-151-RGD) with hyaluronic acid (HA), we have examined the nanostructure, viscosity, friction, and interfacial energy of fluid-fluid phase-separated coacervates using the surface forces apparatus and microscopic techniques. At mixing ratios of fp-151-RGD:HA resulting in marginal coacervation, the coacervates showed shear-thickening viscosity and no structure by cryo-transmission electron microscopy (cryo-TEM). However, at the mixing ratio producing maximum coacervation, the coacervate showed shear-thinning viscosity and a transition to a bicontinuous phase by cryo-TEM. The shear-thinning viscosity, high friction coefficient (>1.2), and low interfacial energy (<1 mJ m(-2)) observed at the optimal mixing ratio for coacervation are promising delivery, spreading and adhesion properties for future wet adhesive and coating technologies.

Development of bioadhesives from marine mussels

Mussel adhesive proteins have received increased attention as potential biomedical and environmentally friendly underwater adhesives thanks to their fascinating properties, including strong and flexible adhesion, adhesion to various material substrates, water displacement, that they are 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, a lot of effort has been put into realizing the promise of these bioadhesive materials from marine mussels. Here, progress in the diverse developmental approaches, with particular emphasis on functional production of mussel adhesive proteins, are reviewed.

Mussel-Mimetic Protein-Based Adhesive Hydrogel

Hydrogel systems based on cross-linked polymeric materials which could provide both adhesion and cohesion in wet environment have been considered as a promising formulation of tissue adhesives. Inspired by marine mussel adhesion, many researchers have tried to exploit the 3,4-dihydroxyphenylalanine (DOPA) molecule as a cross-linking mediator of synthetic polymer-based hydrogels which is known to be able to achieve cohesive hardening as well as adhesive bonding with diverse surfaces. Beside DOPA residue, composition of other amino acid residues and structure of mussel adhesive proteins (MAPs) have also been considered important elements for mussel adhesion. Herein, we represent a novel protein-based hydrogel system using DOPA-containing recombinant MAP. Gelation can be achieved using both oxdiation-induced DOPA quinone-mediated covalent and Fe(3+)-mediated coordinative noncovalent cross-linking. Fe(3+)-mediated hydrogels show deformable and self-healing viscoelastic behavior in rheological analysis, which is also well-reflected in bulk adhesion strength measurement. Quinone-mediated hydrogel has higher cohesive strength and can provide sufficient gelation time for easier handling. Collectively, our newly developed MAP hydrogel can potentially be used as tissue adhesive and sealant for future applications.

Promotion of osteoblast proliferation on complex coacervation-based hyaluronic acid – recombinant mussel adhesive protein coatings on titanium

Many biological polyelectrolytes are capable of undergoing a fluid-fluid phase separation known as complex coacervation. Coacervates were prepared using hyaluronic acid (HA) and a recombinant fusion protein consisting of mussel adhesive motifs and the RGD peptide (fp-151-RGD). The low interfacial energy of the coacervate was exploited to coat titanium (Ti), a metal widely used in implant materials. The coacervate effectively distributed both HA and fp-151-RGD over the Ti surfaces and enhanced osteoblast proliferation. Approximately half of total fp-151-RGD and HA in the solution transferred to the titanium surface within 2h. Titanium coated with coacervates having high residual negative surface charge showed the highest cell proliferation of preosteoblast cells (MC-3T3) compared to the treatments tested. Indeed, MC-3T3 cells on complex coacervate coated titanium foils exhibited over 5 times greater cell proliferation than bare, HA coated or fp-151-RGD coated titanium.

Expression of Functional Recombinant Mussel Adhesive Protein Type 3A in Escherichia coli

Mussel adhesive proteins, including the 20‐plus variants of foot protein type 3 (fp‐3), have been suggested as potential environmentally friendly adhesives for use in aqueous conditions and in medicine. Here we report the novel production of a recombinant Mytilus galloprovincialis foot protein type 3 variant A (Mgfp‐3A) fused with a hexahistidine affinity ligand in Escherichia coli and its ∼99% purification with affinity chromatography. Recombinant Mgfp‐3A showed a superior purification yield and better apparent solubility in 5% acetic acid (prerequisites for large‐scale production and practical use) compared to those of the previously reported recombinant M. galloprovincialis foot protein type 5 (Mgfp‐5). The adsorption abilities and adhesion forces of purified recombinant Mgfp‐3A were compared with those of Cell‐Tak (a commercial mussel extract adhesive) and recombinant Mgfp‐5 using quartz crystal microbalance analysis and modified atomic force microscopy, respectively. These assays showed that the adhesive ability of recombinant Mgfp‐3A was comparable to that of Cell‐Tak but lower than that of recombinant Mgfp‐5. Collectively, these results indicate that recombinant Mgfp‐3A may be useful as a commercial bioadhesive or an adhesive ingredient in medical or underwater environments.

Improved Performance of Protected Catecholic Polysiloxanes for Bioinspired Wet Adhesion to Surface Oxides

A facile synthetic strategy for introducing catecholic moieties into polymeric materials based on a readily available precursor (eugenol) and efficient chemistries [tris(pentafluorophenyl)borane-catalyzed silation and thiol-ene coupling] is reported. Silyl protection is shown to be critical for the oxidative stability of catecholic moieties during synthesis and processing, which allows functionalized polysiloxane derivatives to be fabricated into 3D microstructures as well as 2D patterned surfaces. Deprotection gives stable catechol surfaces whose adhesion to a variety of oxide surfaces can be precisely tuned by the level of catechol incorporation. The advantage of silyl protection for catechol-functionalized polysiloxanes is demonstrated and represents a promising and versatile new platform for underwater surface treatments.