Dong Gyun Kang

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.

Enhanced detoxification of organophosphates using recombinant Escherichia coli with co-expression of organophosphorus hydrolase and bacterial hemoglobin

A significantly improved, recombinant Escherichia coli has been developed to degrade the toxic organophosphorus compound, Paraoxon, to non-toxic materials by co-expression of organophosphorus hydrolase (OPH) under trc promoter and Vitreoscilla hemoglobin (VHb) under O2dependent nar promoter. VHb-expressing whole cells had significant enhancement of OPH activity (48%, 18.7 vs. 27.8 unit l−1) and bioconversion efficiency Vmax/Km (44%, 0.14 vs. 0.2 min−1) compared to VHb-free system.

Enhanced biodegradation of toxic organophosphate compounds using recombinant Escherichia coli with sec pathway-driven periplasmic secretion of organophosphorus hydrolase.

Although Escherichia coli can be genetically engineered to degrade environmental toxic organophosphate compounds (OPs) to nontoxic materials, a critical problem in such whole cell systems is limited substrate diffusion. The present work examined whether periplasmic expression of organophosphorus hydrolase (OPH) resulted in better whole cell enzymatic activity compared to standard cytosolic expression. Recombinant OPH periplasmic expression was achieved using the general secretory (sec) pathway with the pelB signal sequence. We found that while total OPH activity in periplasmic‐expressing cell lysates was lower compared to that in cytosolic‐expressing cell lysates whole cell OPH activity was 1.8‐fold greater at 12 h post‐induction in the periplasmic‐expressing cells as a result of OPH translocation into the periplasmic space (∼67% of whole cell OPH activity was found in the periplasmic fraction). These data suggest that E. coli engineered to periplasmically secrete OPH via the sec pathway may provide an improved whole cell biodegradation system for destruction of environmental toxic OPs.

Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as a Biocatalyst for CO2 Sequestration

ABSTRACT Carbonic anhydrase is an enzyme that reversibly catalyzes the hydration of carbon dioxide (CO2). It has been suggested recently that this remarkably fast enzyme can be used for sequestration of CO2, a major greenhouse gas, making this a promising alternative for chemical CO2 mitigation. To promote the economical use of enzymes, we engineered the carbonic anhydrase from Neisseria gonorrhoeae (ngCA) in the periplasm of Escherichia coli, thereby creating a bacterial whole-cell catalyst. We then investigated the application of this system to CO2 sequestration by mineral carbonation, a process with the potential to store large quantities of CO2. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial cell displayed the distinct ability to hydrate CO2 compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO3) formation and exerted a striking impact on the maximal amount of CaCO3 produced under conditions of relatively low pH. It was also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate that the engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO2 sequestration.

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.

Suppression of β-N-acetylglucosaminidase in the N-glycosylation pathway for complex glycoprotein formation in Drosophila S2 cells

Most insect cells have a simple N-glycosylation process and consequently paucimannosidic or simple core glycans predominate. Previously, we have shown that paucimannosidic N-glycan structures are dominant in Drosophila S2 cells. It has been proposed that beta-N-acetylglucosaminidase (GlcNAcase), a hexosaminidase in the Golgi membrane which removes a terminal N-acetylglucosamine (GlcNAc), might contribute to simple N-glycosylation in several insects and insect-derived cells except S2 cells. In the present work, we investigated the substantial effects of GlcNAcase on N-glycan patterns in Drosophila S2 cells using two GlcNAcase suppression strategies: an mRNA-targeting approach using RNA interference (RNAi) and a protein-targeting approach using the specific chemical inhibitor 2-acetamido-1,2-dideoxynojirimycin (2-ADN). Using high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses, we found that the N-glycosylation patterns of human erythropoietin (hEPO) secreted by stably transfected S2 cells were more complex following GlcNAcase suppression, which generated N-glycan structures with a terminal GlcNAc and/or galactose. These data demonstrate that GlcNAcase may be an important factor in the formation of paucimannosidic core N-glycans in Drosophila S2 cells and suggest that it may be possible to express complex glycoproteins in engineered Drosophila S2 cells by suppressing GlcNAcase in the N-glycosylation pathway.

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.

Enhancement of Mussel Adhesive Protein Production in Escherichia coli by Co‐expression of Bacterial Hemoglobin

Mussel adhesive proteins (MAPs) have been considered as potential underwater and medical bioadhesives. Previously, we reported a functional expression of recombinant MAP hybrid fp‐151, which is a fusion protein with six type 1 (fp‐1) decapeptide repeats at each type 5 (fp‐5) terminus, with practical properties in Escherichia coli. In the present work, we introduced the Vitreoscilla hemoglobin (VHb) co‐expression strategy to enhance the production levels of hybrid fp‐151 since VHb has been successfully used for efficient oxygen utilization in several expression systems, including E. coli. In both batch‐type flask and fed‐batch‐type bioreactor cultures, we found that co‐expression of VHb conferred higher cell growth and hybrid fp‐151 production. Its positive effects were significantly increased in high cell density bioreactor cultures as the microaerobic environment was more quickly and severely formed. We obtained a ∼1.9‐fold higher (∼1 g/L) production of MAP fp‐151 from VHb co‐expressing cells in fed‐batch bioreactor cultures as compared to that from VHb non‐expressing cells. Collectively and regardless of the culture type, VHb co‐expression strategy was successful in enhancing the production of recombinant mussel adhesive proteins in the E. coli expression system.