Byeongseon Yang

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.

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.

A tyrosinase, mTyr-CNK, that is functionally available as a monophenol monooxygenase

Tyrosinase efficiently catalyzes the ortho-hydroxylation of monophenols and the oxidation of diphenols without any additional cofactors. Although it is of significant interest for the biosynthesis of catechol derivatives, the rapid catechol oxidase activity and inactivation of tyrosinase have hampered its practical utilization as a monophenol monooxygenase. Here, we prepared a functional tyrosinase that exhibited a distinguished monophenolase/diphenolase activity ratio (Vmax mono/ Vmax di = 3.83) and enhanced catalytic efficiency against L-tyrosine (kcat = 3.33 ± 0.18 s−1, Km = 2.12 ± 0.14 mM at 20 °C and pH 6.0). This enzyme was still highly active in ice water (>80%), and its activity was well conserved below 30 °C. In vitro DOPA modification, with a remarkably high yield as a monophenol monooxygenase, was achieved by the enzyme taking advantage of these biocatalytic properties. These results demonstrate the strong potential for this enzyme’s use as a monophenol monooxygenase in biomedical and industrial applications.

Self-assembled adhesive biomaterials formed by a genetically designed fusion protein

Here we report a recombinant protein (MS) obtained by genetic fusion of a mussel foot protein (Mfp3) motif into a silk spidroin (MaSp1). The MS not only self-assembled into a supramolecular fibre, as does the parent MaSp1, but also showed enhanced adhesiveness resulting from the DOPA-containing Mfp3 portion. The successful incorporation of the wet adhesiveness of Mfp3 into the well-structured assembly of MaSp1 may provide a new insight for the genetic design of underwater adhesive recombinant proteins by utilizing the structural features of a spidroin protein.

Sprayable Adhesive Nanotherapeutics: Mussel-Protein-Based Nanoparticles for Highly Efficient Locoregional Cancer Therapy

Following surgical resection for primary treatment of solid tumors, systemic chemotherapy is commonly used to eliminate residual cancer cells to prevent tumor recurrence. However, its clinical outcome is often limited due to insufficient local accumulation and the systemic toxicity of anticancer drugs. Here, we propose a sprayable adhesive nanoparticle (NP)-based drug delivery system using a bioengineered mussel adhesive protein (MAP) for effective locoregional cancer therapy. The MAP NPs could be administered to target surfaces in a surface-independent manner through a simple and easy spray process by virtue of their unique adhesion ability and sufficient dispersion property. Doxorubicin (DOX)-loaded MAP NPs (MAP@DOX NPs) exhibited efficient cellular uptake, endolysosomal trafficking, and subsequent low pH microenvironment-induced DOX release in cancer cells. The locally sprayed MAP@DOX NPs showed a significant inhibition of tumor growth in vivo, resulting from the prolonged retention of the MAP@DOX NPs on the tumor surface. Thus, this adhesive MAP NP-based spray therapeutic system provides a promising approach for topical drug delivery in adjuvant cancer therapy.

Mussel-Derived Bioadhesives

Marine mussels use MAPs (MAPs) for their adhesion. MAPs have fascinating properties, including strong adhesion to various material substrates, water displacement, biocompatibility, and controlled biodegradability. In this work, among six types of MAPs, including fp-1–fp-6, biosynthetic constructs of MAPs are considered; hybrid type recombinant MAPs are designed to improve productivity and purification. Hybrid recombinant fp-151, which comprises six decapeptide repeats of fp-1 at both N and C-termini of fp-5, was successfully overexpressed in a bacterial system, showing approximately ≈ 1 g / L Open image in new window production yield in a pilot scale fed-batch bioreactor culture. For industrial applications, it was attempted to use MAPs in tissue engineering fields as coating extracellular matrix (ECM ) through surface modification and constructing nanofibrous scaffolds. As a result, the MAP-based coating strategy could be generally applied for facile and efficient surface modification of negatively charged bioactive molecules for tissue engineering. The use of MAP-based nanofibers could provide bioactive peptides efficiently onto the scaffold surface, enhancing the cell attachment and proliferation on the nanofibers fabricated using RGD peptide-conjugated MAPs compared with bare polycaprolactone (PCL ) polymer nanofibers as well having a four times higher mechanical strength. Also, easy fabrication through blending with diverse types of synthetic polymers and significant bone regeneration was observed. In addition, there was a trial for utilization of MAPs in pharmaceutics, cosmetics, and food industries with encapsulating active molecules such as chemical drugs, proteins, cells, and flavor ingredients through a complex coacervation technique based on MAPs.