Yoo Seong Choi

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.

Biomineralization-based conversion of carbon dioxide to calcium carbonate using recombinant carbonic anhydrase

Recently, as a mimic of the natural biomineralization process, the use of carbonic anhydrase (CA), which is an enzyme catalyzing fast reversible hydration of carbon dioxide to bicarbonate, has been suggested for biological conversion of CO(2) to valuable chemicals. While purified bovine CA (BCA) has been used in previous studies, its practical utilization in CO(2) conversion has been limited due to the expense of BCA preparation. In the present work, we investigated conversion of CO(2) into calcium carbonate as a target carbonate mineral by using a more economical, recombinant CA. To our knowledge, this is the first report of the usage of recombinant CA for biological CO(2) conversion. Recombinant α-type CA originating in Neisseria gonorrhoeae (NCA) was highly expressed as a soluble form in Escherichia coli. We found that purified recombinant NCA which showed comparable CO(2) hydration activity to commercial BCA significantly promoted formation of solid CaCO(3) through the acceleration of CO(2) hydration rate, which is naturally slow. In addition, the rate of calcite crystal formation was also accelerated using recombinant NCA. Moreover, non-purified crude recombinant NCA also showed relatively significant ability. Therefore, recombinant CA could be an effective, economical biocatalyst in practical CO(2) conversion system.

Recombinant mussel adhesive protein fp-5 (MAP fp-5) as a bulk bioadhesive and surface coating material

Mussel adhesive proteins (MAPs) attach to all types of inorganic and organic surfaces, even in wet environments. MAP of type 5 (fp-5), in particular, has been considered as a key adhesive material. However, the low availability of fp-5 has hampered its biochemical characterization and practical applications. Here, soluble recombinant fp-5 is mass-produced in Escherichia coli. Tyrosinase-modified recombinant fp-5 showed ∼1.11 MPa adhesive shear strength, which is the first report of a bulk-scale adhesive force measurement for purified recombinant of natural MAP type. Surface coatings were also performed through simple dip-coating of various objects. In addition, complex coacervate using recombinant fp-5 and hyaluronic acid was prepared as an efficient adhesive formulation, which greatly improved the bulk adhesive strength. Collectively, it is expected that this work will enhance basic understanding of mussel adhesion and that recombinant fp-5 can be successfully used as a realistic bulk-scale bioadhesive and an efficient surface coating material.

In Vivo Residue‐Specific Dopa‐Incorporated Engineered Mussel Bioglue with Enhanced Adhesion and Water Resistance

Misaminoacylation of 3,4-dihydroxyphenylalanine (Dopa) molecules to tRNA(Tyr) by endogenous tyrosyl-tRNA synthetase allowed the quantitative replacement of tyrosine residues with a yield of over 90 % by an in vivo residue-specific incorporation strategy, to create, for the first time, engineered mussel adhesive proteins (MAPs) in Escherichia coli with a very high Dopa content, close to that of natural MAPs. The Dopa-incorporated MAPs exhibited a superior surface adhesion and water resistance ability by assistance of Dopa-mediated interactions including the oxidative Dopa cross-linking, and furthermore, showed underwater adhesive properties comparable to those of natural MAPs. These results propose promising use of Dopa-incorporated engineered MAPs as bioglues or adhesive hydrogels for practical underwater applications.

In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli

BackgroundIn nature, mussel adhesive proteins (MAPs) show remarkable adhesive properties, biocompatibility, and biodegradability. Thus, they have been considered promising adhesive biomaterials for various biomedical and industrial applications. However, limited production of natural MAPs has hampered their practical applications. Recombinant production in bacterial cells could be one alternative to obtain useable amounts of MAPs, although additional post-translational modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (Dopa) and Dopaquinone is required. The superior properties of MAPs are mainly attributed to the introduction of quinone-derived intermolecular cross-links. To solve this problem, we utilized a co-expression strategy of recombinant MAP and tyrosinase in Escherichia coli to successfully modify tyrosine residues in vivo.ResultsA recombinant hybrid MAP, fp-151, was used as a target for in vivo modification, and a dual vector system of pET and pACYC-Duet provided co-expression of fp-151 and tyrosinase. As a result, fp-151 was over-expressed and mainly obtained from the soluble fraction in the co-expression system. Without tyrosinase co-expression, fp-151 was over-expressed in an insoluble form in inclusion bodies. The modification of tyrosine residues in the soluble-expressed fp-151 was clearly observed from nitroblue tetrazolium staining and liquid-chromatography-mass/mass spectrometry analyses. The purified, in vivo modified, fp-151 from the co-expression system showed approximately 4-fold higher bulk-scale adhesive strength compared to in vitro tyrosinase-treated fp-151.ConclusionHere, we reported a co-expression system to obtain in vivo modified MAP; additional in vitro tyrosinase modification was not needed to obtain adhesive properties and the in vivo modified MAP showed superior adhesive strength compared to in vitro modified protein. It is expected that this co-expression strategy will accelerate the use of functional MAPs in practical applications and can be successfully applied to prepare other Dopa/Dopaquinone-based biomaterials.

Reinforced Multifunctionalized Nanofibrous Scaffolds Using Mussel Adhesive Proteins

Nanofiber technology has been largely recognized because of its ability to fabricate multiple nanosized fibers that are structurally similar to collagen fibrils of the natural extracellular matrix (ECM). The large surface area of nanofibers is a major advantage of nanofibrous scaffolds because these are the sites at which cell–substrate interactions occur. Rendering the nanofiber surface to be biofunctional is critical for its successful application in tissue engineering. Synthetic and natural biomaterials have been widely used for the fabrication of nanofibrous scaffolds for tissue engineering applications. Blending synthetic and natural biomaterials has been used to combine the excellent biological activities of natural biomaterials with the high processability and desired mechanical strengths of synthetic polymers; however, this approach has had limited success for several pairs of synthetic and natural biomaterials. Several bioactive molecules, such as ECM proteins, ECM carbohydrates, ECM-derived peptides, and growth factors, have been introduced onto the surface of nanofibers to design biofunctional and biomimetic tissue engineering scaffolds. Generally, typical conjugation chemistry and physical adsorption procedures have been used to immobilize biomolecules after the completion of the surface activation steps, including plasma and wet-chemical treatments or surface graft polymerization. However, these strategies are limited in availability because of the multiple, complicated procedures that are required to complete them. Thus, development of a simple and effective surface functionalization strategy for nanofibers would be a promising tool for successful tissue engineering applications. In the present work, we propose to use mussel adhesive protein (MAP) as a natural biomaterial that serves as a blending partner for the preparation of sticky nanofibrous scaffolds. Using MAPs as a blending partner provides a facile, efficient, and multifunctionalizing platform for generating novel nanofibrous scaffolds. In fact, nanofibrous scaffolds based on MAPs having mechanical reinforcement were fabricated by a simple e lectrospinning process to develop a “general coating platform” for diverse bioactive molecules (Figure 1).

In vivo post-translational modifications of recombinant mussel adhesive protein in insect cells†

Mussel adhesive proteins (MAPs) have been suggested as promising bioadhesives for diverse application fields, including medical uses. Previously, we successfully constructed and produced a new type of functional recombinant MAP, fp‐151, in a prokaryotic Escherichia coli expression system. Even though the E. coli‐derived MAP showed several excellent features, such as high production yield and efficient purification, in vitro enzymatic modification is required to convert tyrosine residues to l‐3,4‐dihydroxyphenyl alanine (dopa) molecules for its adhesive ability, due to the intrinsic inability of E. coli to undergo post‐translational modification. In this work, we produced a soluble recombinant MAP in insect Sf9 cells, which are widely used as an effective and convenient eukaryotic expression system for eukaryotic foreign proteins. Importantly, we found that insect‐derived MAP contained converted dopa residues by in vivo post‐translational modification. In addition, insect‐derived MAP also had other post‐translational modifications including phosphorylation of serine and hydroxylation of proline that originally occurred in some natural MAPs. To our knowledge, this is the first report on in vivo post‐translational modifications of MAP containing dopa and other modified amino acid residues.

A comparative study on the bulk adhesive strength of the recombinant mussel adhesive protein fp-3

Mussel adhesive protein (MAP) type 3 (fp-3) is considered one of the key components for mussel adhesion. However, its bulk adhesive strength has not been characterized due to its availability in limited quantities. In the present work, a feasible production (∼47 mg l−1) of recombinant fp-3 was achieved, and its bulk adhesive strength was measured for the first time; ∼0.57 MPa for the unmodified form and ∼0.94 and ∼2.28 MPa for the 3,4-dihydroxy-L-phenylalanine (DOPA)-modified form, having a 9.6% yield without and with oxidant treatment, respectively. Furthermore, values for the bulk adhesive strength of several DOPA-modified recombinant MAPs were compared. The maximum adhesive strength of DOPA-modified fp-3 after oxidant treatment was stronger than that of type 5 (fp-5), which has a 6.2% modification yield, and was comparable to that of hybrid types fp-131 and fp-151, which have similar yields (∼5%). The strong bulk adhesive property of recombinant fp-3 demonstrates its potential use as a promising bioadhesive.

High expression and biosilica encapsulation of alkaline-active carbonic anhydrase for CO2 sequestration system development

Carbonic anhydrase (CA) is a biocatalyst for CO2 sequestration because of its distinctive ability to accelerate CO2 hydration. High production and efficient immobilization of alkaline-active CAs are required, because one potential application of CA is its use in the alkaline solvent-based CO2 absorption/desorption process. Here, we designed and applied an α-type CA from Hahella chejuensis (HCA), which was reported as highly active in alkaline conditions, but was mostly expressed as insoluble forms. We found that the signal peptide-removed form of HCA [HCA(SP-)] was successfully expressed in the soluble form [∼70mg of purified HCA(SP-) per L of culture]. HCA(SP-) also displayed high pH stability in alkaline conditions, with maximal activity at pH 10; at this pH, ∼90% activity was maintained for 2h. Then, we prepared HCA(SP-)-encapsulated silica particles [HCA(SP-)@silica] via a spermine-mediated bio-inspired silicification method. HCA(SP-)@silica exhibited high-loading and highly stable CA activity. In addition, HCA(SP-)@silica retained more than 90% of the CA activity even after 10 cycles of use in mild conditions, and ∼80% in pH 10 conditions. These results will be useful for the development of practical CO2 sequestration processes employing CA.