Young Hoon Song

The adhesive properties of coacervated recombinant hybrid mussel adhesive proteins

Marine mussels attach to substrates using adhesive proteins. It has been suggested that complex coacervation (liquid-liquid phase separation via concentration) might be involved in the highly condensed and non-water dispersed adhesion process of mussel adhesive proteins (MAPs). However, as purified natural MAPs are difficult to obtain, it has not been possible to experimentally validate the coacervation model. In the present work, we demonstrate complex coacervation in a system including recombinant MAPs and hyaluronic acid (HA). Our recombinant hybrid MAPs, fp-151 and fp-131, can be produced in large quantities, and are readily purified. We observed successful complex coacervation using cationic fp-151 or fp-131, and an anionic HA partner. Importantly, we found that highly condensed complex coacervates significantly increased the bulk adhesive strength of MAPs in both dry and wet environments. In addition, oil droplets were successfully engulfed using a MAP-based interfacial coacervation process, to form microencapsulated particles. Collectively, our results indicate that a complex coacervation system based on MAPs shows superior adhesive properties, combined with additional valuable features including liquid/liquid phase separation and appropriate viscoelasticity. Our microencapsulation system could be useful in the development of new adhesive biomaterials, including self-adhesive microencapsulated drug carriers, for use in biotechnological and biomedical applications.

Cell behavior on extracellular matrix mimic materials based on mussel adhesive protein fused with functional peptides

Adhesion of cells to surfaces is a basic and important requirement in cell culture and tissue engineering. Here, we designed artificial extracellular matrix (ECM) mimics for efficient cellular attachment, based on mussel adhesive protein (MAP) fusion with biofunctional peptides originating from ECM materials, including fibronectin, laminin, and collagen. Cellular behaviors, including attachment, proliferation, spreading, viability, and differentiation, were investigated with the artificial ECM material-coated surfaces, using three mammalian cell lines (pre-osteoblast, chondrocyte, and pre-adipocyte). All cell lines examined displayed superior attachment, proliferation, spreading, and survival properties on the MAP-based ECM mimics, compared to other commercially available cell adhesion materials, such as poly-L-lysine and the naturally extracted MAP mixture. Additionally, the degree of differentiation of pre-osteoblast cells on MAP-based ECM mimics was increased. These results collectively demonstrate that the artificial ECM mimics developed in the present work are effective cell adhesion materials. Moreover, we expect that the MAP peptide fusion approach can be extended to other functional tissue-specific motifs.

Production of fusion mussel adhesive fp‐353 in Escherichia coli

Mussel adhesive proteins (MAPs) have a potential as environmentally friendly adhesives for use under aqueous conditions. MAPs maybe of particular value in medical applications. We previously reported the functional expression of recombinant foot protein type 5 (fp‐5) and foot protein type 3A (fp‐3A), both of which have significant adhesion abilities, in Escherichia coli. However, these proteins were produced at low levels because of post‐induction cell growth inhibition, and the proteins showed poor post‐purification solubility. Here, we design and produce a new type of recombinant MAP, fp‐353, that is a fusion protein with fp‐3A at each terminus of fp‐5. Because fp‐353 formed inclusion bodies, host cell growth inhibition did not occur. In addition, the solubility of MAP fp‐353 after purification was significantly enhanced, permitting the preparation of a viscous concentrated glue solution for large‐scale adhesion strength measurements. Together with large‐scale cowhide adhesion measurements and cell‐adhesion analyses, we successfully demonstrated that fusion mussel protein fp‐353 has potential as a practical alternative bioadhesive.

Mussel adhesive protein fused with cell adhesion recognition motif triggers integrin-mediated adhesion and signaling for enhanced cell spreading, proliferation, and survival

Adhesion of cells to a surface is a basic and important requirement in the fields of cell culture and tissue engineering. Previously, we constructed the cell adhesive, fp-151-RGD, by fusion of the hybrid mussel adhesive protein, fp-151, and GRGDSP peptide, one of the major cell adhesion recognition motifs; fp-151-RGD efficiently immobilized cells on coated culture surfaces with no protein and surface modifications, and apparently enhanced cell adhesion, proliferation, and spreading abilities. In the present study, we investigated the potential use of fp-151-RGD as a biomimetic extracellular matrix material at the molecular level by elucidating its substantial effects on integrin-mediated adhesion and signaling. Apoptosis derived from serum deprivation was significantly suppressed on the fp-151-RGD-coated surface, indicating that RGD-induced activation of integrin-mediated signaling triggers the pathway for cell survival. Analysis of the phosphorylation of focal adhesion kinase clearly demonstrated activation of focal adhesion kinase, a well-established indicator of integrin-mediated signaling, on the fp-151-RGD-coated surface, leading to significantly enhanced cell behaviors, including proliferation, spreading and survival, and consequently, more efficient cell culture.

Salt Effects on Aggregation and Adsorption Characteristics of Recombinant Mussel Adhesive Protein fp-151

Mussel adhesive proteins (MAPs) are excellent bioadhesives. Although the role of L-3,4-dihydroxyphenyl alanine (DOPA) in water-resistant strong adhesion has been extensively studied, MAP aggregate formation and physical adsorption behavior have not been thoroughly explored. Here, we investigated the aggregation and attachment properties of a recombinant MAP with no DOPA residues. We found that chemical cross-linking of oxidized DOPA residues was not required for aggregate formation; increased anionic kosmotropy and cationic chaotropy allowed aggregation permitting strong adsorption onto hydrophilic surfaces. This study suggests that the initial step in MAP adhesion might thus be aggregation. Physical adsorption can be adjusted by varying the salt ions in water, to mediate initial efficient attachment followed by DOPA-mediated water-resistant strong adhesion.

Recombinant mussel proximal thread matrix protein promotes osteoblast cell adhesion and proliferation

Backgroundvon Willebrand factor (VWF) is a key load bearing domain for mamalian cell adhesion by binding various macromolecular ligands in extracellular matrix such as, collagens, elastin, and glycosaminoglycans. Interestingly, vWF like domains are also commonly found in load bearing systems of marine organisms such as in underwater adhesive of mussel and sea star, and nacre of marine abalone, and play a critical load bearing function. Recently, Proximal Thread Matrix Protein1 (PTMP1) in mussel composed of two vWF type A like domains has characterized and it is known to bind both mussel collagens and mammalian collagens.ResultsHere, we cloned and mass produced a recombinant PTMP1 from E. coli system after switching all the minor codons to the major codons of E. coli. Recombinant PTMP1 has an ability to enhance mouse osteoblast cell adhesion, spreading, and cell proliferation. In addition, PTMP1 showed vWF-like properties as promoting collagen expression as well as binding to collagen type I, subsequently enhanced cell viability. Consequently, we found that recombinant PTMP1 acts as a vWF domain by mediating cell adhesion, spreading, proliferation, and formation of actin cytoskeleton.ConclusionsThis study suggests that both mammalian cell adhesion and marine underwater adhesion exploits a strong vWF-collagen interaction for successful wet adhesion. In addition, vWF like domains containing proteins including PTMP1 have a great potential for tissue engineering and the development of biomedical adhesives as a component for extra-cellular matrix.

Recombinant Mussel Coating Protein Fused with Cell Adhesion Recognition Motif Enhanced Cell Proliferation

Endurable coating on biomedical substrates is one of the most important issues in biomedical engineering field. Recently, a robust yet reversible coating has found in the cuticle of mussel byssus and the mimicking of the mussel cuticle coating has been considered as a strategy to build up endurable coating on biomedical materials. To date, the only known protein in the cuticle is mussel foot protein-1 (fp-1). To form endurable and bioactive coating on the biomedical surface, the fusion protein of fp-1 and GRGDSP peptide (fp-1-RGD) was genetically designed and produced in E. coli. The fusion protein of fp-1-RGD was successfully expressed as a form of inclusion body and was simply purified by diluted acetic acid extraction with high purity (~95%). Fp-1-RGD was coated on the tissue culture polystyrene (TCPS) and showed better preosteoblast cell proliferation than that of TCPS. Therefore, the marriage of fp-1 and bioactive peptide can be a good strategy to form bioactive and endurable coating in biomedical field.

Noninvasive monitoring of environmental toxicity through green fluorescent protein expressing Escherichia coli

The facile and rapid monitoring of the cellular response to environmental stresses is crucial for understanding the effects of environmental toxicity in living organisms. The overall cell growth can be examined to find a simple monitoring system. Green fluorescent protein (GFP) is advantageous when used as a reporter protein of the cellular stress responses in Escherichia coli because it allows the non-invasive monitoring of GFP in vivo without affecting the cell metabolism. We compared the environmental toxicities of chemical pollutants using GFP expressing E. coli for easy monitoring by incubation in various concentrations of harmful chemicals (ethanol, phenol, para-formaldehyde, paraben, and triclosan). The results showed that all the chemical pollutants act on cell growth and the cell metabolism according to the measured cell density and fluorescent intensity of GFP. In addition, from comparative analysis for quantification, the concentration of unknown ethanol toxicity, which was not determined at that concentration, could be deduced. In conclusion, the degree of toxicity for each chemical pollutant could be estimated or evaluated. This system will be useful for monitoring the toxicity of chemical pollutants in a non-invasive monitoring system.

Effect of Culture Conditions on the Whole Cell Activity of Recombinant Escherichia coli Expressing Periplasmic Organophosphorus Hydrolase and Cytosolic GroEL/ES Chaperone

Specific whole cell activity strongly affects sensitivity and detection limit of whole cell-based biosensors. Previously, we developed recombinant Escherichia coli coexpressing periplasmic organophosphorus hydrolase (OPH) and cytosolic chaperone GroEL-GroES (GroEL/ES). In present work, we investigated the effect of culture conditions on whole cell OPH activity. Especially, the whole cell OPH activity was significantly affected by the concentration of tetracycline that is an inducer for chaperone GroEL/ES. When cultured at 20°C for 31 h in M9 medium containing 1 mM IPTG, 50 ng/mL tetracycline, and 500 µM CoCl2, the recombinant E. coli exhibited a specific whole cell OPH activity (U/OD600) of ~0.55, which is 2.6-fold higher than that of recombinant E. coli cultured as previously described conditions. In addition, recombinant cells showed adequate storage stability for 1 week with 100% of original response. Finally, the improved activity and adequate stability in the whole cell biocatalyst will contribute to sensitivity, detection time, and stability of a whole cell-based biosensor for the detection of toxic organophosphates.

Sucrose-calcium Complexation for the Durable Biomass Pellet

Due to their environmental friendliness, woodbased biomass pellets are widely used as conventional fuel sources in daily life and in various industries. Durability and proper mechanical strength are important quality factors for practical applications of biomass pellets as they enable their easy handling, transportation, and storage. In the present study, to increase mechanical strength of sawdust biomass pellet fuels, sucrose was employed as a main binder material. In addition, calcium ions were included as cross-linkers, which improved capability of modified binders. Even though sucrose alone enabled production of pellets with comparatively high compressive strength, addition of several calcium-containing substances further improved mechanical properties of sawdust pellets. Interestingly, we found that a combination of sucrose with CaCl2 (acidic blended solution) decreased, whereas addition of CaO or Ca(OH)2 (basic blended solution) considerably enhanced pellet strength. Thus, we concluded that calcium ions are able to form stable complexes with sucrose at basic pH levels (>10). Therefore, materials incorporating sucrose-calcium complexes can be successfully used as eco-friendly novel binders for the construction of durable biomass pellet fuels. Furthermore, their applications can be extended to formulations of nutritional or pharmaceutical substances.