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Hyung Joon Cha - One of the best experts on this subject based on the ideXlab platform.
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enhanced production of dopa incorporated Mussel Adhesive Protein using engineered translational machineries
Biotechnology and Bioengineering, 2020Co-Authors: Ye Seul Jeong, Hyung Joon Cha, Byeongseon Yang, Byungseop Yang, Mincheol Shin, Jihyoun Seong, Inchan KwonAbstract:Mussel Adhesive Proteins (MAPs) have great potential as bioglues, particularly in wet conditions. Although in vivo residue-specific incorporation of 3,4-dihydroxyphenylalanine (Dopa) in tyrosine-auxotrophic Escherichia coli cells allows for production of Dopa-incorporated bioengineered MAPs (dMAPs), the low production yield hinders the practical application of dMAPs. This low production yield of dMAPs is due to low translational activity of a noncanonical amino acid, Dopa, in E. coli cells. Herein, to enhance the production yield of dMAPs, we investigated the coexpression of Dopa-recognizing tyrosyl-tRNA synthetases (TyrRSs). To use the Dopa-specific Methanococcus jannaschii TyrRS (MjTyrRS-Dopa), we altered the anticodon of tyrosyl-tRNA amber suppressor into AUA (MjtRNATyr AUA ) to recognize a tyrosine codon (AUA). Co-overexpression of MjTyrRS-Dopa and MjtRNATyr AUA increased the production yield of Dopa-incorporated MAP foot Protein type 3 (dfp-3) by 57%. Similarly, overexpression of E. coli TyrRS (EcTyrRS) led to a 72% higher production yield of dfp-3. Even with coexpression of Dopa-recognizing TyrRSs, dfp-3 has a high Dopa incorporation yield (over 90%) compared to ones prepared without TyrRS coexpression.
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Enhanced production of Dopa‐incorporated Mussel Adhesive Protein using engineered translational machineries
Biotechnology and bioengineering, 2020Co-Authors: Ye Seul Jeong, Hyung Joon Cha, Byeongseon Yang, Byungseop Yang, Mincheol Shin, Jihyoun Seong, Inchan KwonAbstract:Mussel Adhesive Proteins (MAPs) have great potential as bioglues, particularly in wet conditions. Although in vivo residue-specific incorporation of 3,4-dihydroxyphenylalanine (Dopa) in tyrosine-auxotrophic Escherichia coli cells allows for production of Dopa-incorporated bioengineered MAPs (dMAPs), the low production yield hinders the practical application of dMAPs. This low production yield of dMAPs is due to low translational activity of a noncanonical amino acid, Dopa, in E. coli cells. Herein, to enhance the production yield of dMAPs, we investigated the coexpression of Dopa-recognizing tyrosyl-tRNA synthetases (TyrRSs). To use the Dopa-specific Methanococcus jannaschii TyrRS (MjTyrRS-Dopa), we altered the anticodon of tyrosyl-tRNA amber suppressor into AUA (MjtRNATyr AUA ) to recognize a tyrosine codon (AUA). Co-overexpression of MjTyrRS-Dopa and MjtRNATyr AUA increased the production yield of Dopa-incorporated MAP foot Protein type 3 (dfp-3) by 57%. Similarly, overexpression of E. coli TyrRS (EcTyrRS) led to a 72% higher production yield of dfp-3. Even with coexpression of Dopa-recognizing TyrRSs, dfp-3 has a high Dopa incorporation yield (over 90%) compared to ones prepared without TyrRS coexpression.
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Facile Surface Functionalization with Glycosaminoglycans by Direct Coating with Mussel Adhesive Protein
'Mary Ann Liebert Inc', 2018Co-Authors: Bh Choi, Ds Hwang, Yoo Sung Choi, Hyung Joon ChaAbstract:The use of Mussel Adhesive Proteins (MAPs) as a surface coating for cell adhesion has been suggested due to their unique properties of biocompatibility and effective adhesion on diverse inorganic and organic surfaces. The surface functionalization of scaffolds or implants using extracellular matrix (ECM) molecules is important for the enhancement of target cell behaviors such as proliferation and differentiation. In the present work, we suggest a new, simple surface functionalization platform based on the charge interactions between the positively charged MAP linker and negatively charged ECM molecules, such as glycosaminoglycans (GAGs). MAP was efficiently coated onto a titanium model surface using its adhesion ability. Then, several GAG molecules, including hyaluronic acid (HA), heparin sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS), were effectively immobilized on the MAP-coated surfaces by charge interactions. Using HA as a model GAG molecule, we found that the proliferation, spreading, and differentiation behaviors of mouse preosteoblast cells were all significantly improved on MAP/HA-layered titanium. In addition, we successfully constructed a multilayer film on a titanium surface with oppositely charged layer-by-layer coatings of MAP and HA. Collectively, our simple MAP-based surface functionalization strategy can be successfully used for the efficient surface immobilization of negatively charged ECM molecules in various tissue engineering and medical implantation applications.X1131
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Mussel Adhesive Protein-based whole cell array biosensor for detection of organophosphorus compounds
'Elsevier BV', 2018Co-Authors: Cs Kim, Bong-hyuk Choi, Jeong Hyun Seo, Lim G, Hyung Joon ChaAbstract:A whole cell array biosensor for the efficient detection of neurotoxic organophosphate compounds (OPs) was developed through the immobilization of recombinant Escherichia coli cells containing periplasmic-expressing organophosphorus hydrolase (OPH) onto the surface of a 96-well microplate using Mussel Adhesive Protein (MAP) as a microbial cell-immobilizing linker. Both the paraoxon-hydrolyzing activity and fluorescence microscopy analyses demonstrated that the use of MAP in a whole cell biosensor increased the cell-immobilizing efficiency and enhanced the stability of immobilized cells compared to a simple physical adsorption-based whole cell system. Scanning electron microscopic analyses also showed that the E. coli cells were effectively immobilized on the MAP-coated surface without any pretreatment steps. The whole cell array biosensor system, prepared using optimal MAP coating (50 mu g/cm(2)) and cell loading (4 OD600), detected paraoxon levels as low as 5 mu M with high reproducibility, and its quantitative detection range was similar to 5-320 mu M. The MAP-based whole cell array biosensor showed a good long-term stability for 28 day with 80% retained activity and a reusability of up to 20 times. In addition, paraoxon in tap water was also successfully detected without a reduction in sensitivity. Our results indicate that the proposed MAP-based whole cell array system could be used as a potential platform for a stable and reusable whole cell biosensor. (C) 2012 Elsevier B.V. All rights reserved.X1202
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Accelerated skin wound healing using electrospun nanofibrous mats blended with Mussel Adhesive Protein and polycaprolactone
Journal of biomedical materials research. Part A, 2016Co-Authors: Bum Jin Kim, Bong-hyuk Choi, Hogyun Cheong, Eun-som Choi, So-hee Yun, Ki-soo Park, Ick Soo Kim, Dae-hwan Park, Hyung Joon ChaAbstract:Nanofibrous scaffolds have been assessed as one of many promising tissue engineering scaffolds to be utilized for wound-healing applications. Previously, we reported multi-functionalized electrospun nanofibrous scaffolds blended with Mussel Adhesive Protein (MAP) and polycaprolactone (PCL), which provide durable mechanical strength, cell-friendly environments, and a substantial ability to capture diverse bioactive molecules without any surface modifications. In the present work, we applied the blended nanofibrous mats of MAP and PCL for in vivo skin wound healing. The nanofibrous mats showed accelerated regeneration in a rat skin wound-healing model, which might be attributed to a highly compatible environment for keratinocyte cell growth, an ability to capture inherent growth factors, and an efficient exudate absorption capacity. Thus, this work would suggest that Adhesive property of scaffold could be a factor of successful application for wound healing. The MAP-blended nanofibers could also be potentially exploited for diverse tissue regeneration applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 218–225, 2017.
Seonghye Lim - One of the best experts on this subject based on the ideXlab platform.
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RECOMBINANT Mussel Adhesive Protein AS A GENE DELIVERY MATERIAL
'Wiley', 2019Co-Authors: Ds Hwang, Yoo Seong Choi, Kyoung Ro Kim, Seonghye Lim, Cha, Hyung JoonAbstract:Efficient target gene delivery into eukaryotic cells is important for biotechnological research and gene therapy. Gene delivery based on Proteins, including histones, has recently emerged as a powerful non-viral DNA transfer technique. Here, we investigated the potential use of a recombinant Mussel Adhesive Protein, hybrid fp-151, as a gene delivery material, in view of its similar basic amino acid composition to histone Proteins, and cost-effective and high-level production in Escherichia coli. After confirming DNA binding affinity, we transfected mammalian cells (human 293T and mouse NIH/3T3) with foreign genes using hybrid fp-151 as the gene delivery carrier. Hybrid fp-151 displayed comparable transfection efficiency in both mammalian cell lines, compared to the widely used transfection agent, Lipofectamine (TM) 2000. Our results indicate that this Mussel Adhesive Protein may be used as a potential Protein-based gene-transfer mediator.Efficient target gene delivery into eukaryotic cells is important for biotechnological research and gene therapy. Gene delivery based on Proteins, including histones, has recently emerged as a powerful non-viral DNA transfer technique. Here, we investigated the potential use of a recombinant Mussel Adhesive Protein, hybrid fp-151, as a gene delivery material, in view of its similar basic amino acid composition to histone Proteins, and cost-effective and high-level production in Escherichia coli. After confirming DNA binding affinity, we transfected mammalian cells (human 293T and mouse NIH/3T3) with foreign genes using hybrid fp-151 as the gene delivery carrier. Hybrid fp-151 displayed comparable transfection efficiency in both mammalian cell lines, compared to the widely used transfection agent, Lipofectamine (TM) 2000. Our results indicate that this Mussel Adhesive Protein may be used as a potential Protein-based gene-transfer mediator.X1
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Bulk Adhesive strength of recombinant hybrid Mussel Adhesive Protein
'Informa UK Limited', 2018Co-Authors: Hj Cha, Ds Hwang, Seonghye Lim, James D. White, Cristina R. Matos-pérez, Jonathan J. WilkerAbstract:Mussel Adhesive Proteins (MAPs) have received increased attention as potential biomedical and environmental friendly Adhesives. However, practical application of MAPs has been severely limited by uneconomical extraction and unsuccessful genetic production. Developing new Adhesives requires access to large quantities of material and demonstrations of bulk mechanical properties. Previously, the authors designed fp-151, a fusion Protein comprised of six MAP type 1 (fp-1) decapeptide repeats at each MAP type 5 (fp-5) terminus and successfully expressed it in Escherichia coli. This recombinant hybrid Protein exhibited high-level expression, a simple purification and high biocompatibility as well as strong Adhesive ability on a micro-scale. In the present work, investigations on the bulk Adhesive properties of semi-purified (90% purity) fusion fp-151 were performed in air. The unmodified recombinant fp-151, as expressed, contains tyrosine residues and showed significant shear-Adhesive forces (0.33MPa). Adhesion strength increased (0.45MPa) after enzymatic oxidation of tyrosine residues to l-3,4-dihydroxyphenylalanine (DOPA) groups. Addition of cross-linkers such as iron(III), manganese(III) and periodate (IO4-) generally enhanced adhesion, although too much addition decreased adhesion. Among the three cross-linking reagents examined, the non-metallic oxidant periodate showed the highest shear-Adhesive forces (0.86MPa). In addition, it was found that Adhesive strengths could be increased by adding weights to the samples. The highest adhesion strength found was that of DOPA-containing fp-151 cross-linked with periodate and having weights applied to the samples (1.06MPa). Taken together, the first bulk-scale Adhesive force measurements are presented for an expressed recombinant hybrid Mussel Adhesive Protein.X1393
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Interfacial Tension of Complex Coacervated Mussel Adhesive Protein According to the Hofmeister Series
Langmuir : the ACS journal of surfaces and colloids, 2014Co-Authors: Seonghye Lim, Jeong Hyun Seo, Dustin Moon, Hyo Jeong Kim, In Seok Kang, Hyung Joon ChaAbstract:Complex coacervation is a liquid–liquid phase separation in a colloidal system of two oppositely charged polyelectrolytes or colloids. The interfacial tension of the coacervate phase is the key parameter for micelle formation and interactions with the encapsulating material. However, the relationship between interfacial tensions and various salt solutions is poorly understood in complex coacervation. In the present work, the complex coacervate dynamics of recombinant Mussel Adhesive Protein (MAP) with hyaluronic acid (HA) were determined in the presence of Hofmeister series salt ions. Using measurements of absorbance, hydrodynamic diameter, capillary force, and receding contact angle in the bulk phase, the interfacial tensions of complex coacervated MAP/HA were determined to be 0.236, 0.256, and 0.287 mN/m in 250 mM NaHCOO, NaCl, and NaNO3 solutions, respectively. The sequences of interfacial tensions and contact angles of the complex coacervates in the presence of three sodium salts with different anions...
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in vivo post translational modifications of recombinant Mussel Adhesive Protein in insect cells
Biotechnology Progress, 2011Co-Authors: Seonghye Lim, Yoo Seong Choi, Kyoung Ro Kim, Dae-kyum Kim, Daehee Hwang, Hyung Joon ChaAbstract: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.
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In vivo post‐translational modifications of recombinant Mussel Adhesive Protein in insect cells
Biotechnology progress, 2011Co-Authors: Seonghye Lim, Yoo Seong Choi, Kyoung Ro Kim, Dae-kyum Kim, Daehee Hwang, Hyung Joon ChaAbstract: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.
Dong Soo Hwang - One of the best experts on this subject based on the ideXlab platform.
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cation π interaction in dopa deficient Mussel Adhesive Protein mfp 1
Journal of Materials Chemistry B, 2015Co-Authors: Sangsik Kim, Ali Faghihnejad, Yongjin Lee, Yongseok Jho, Hongbo Zeng, Dong Soo HwangAbstract:Here we report the possible contribution of cation–π interaction to underwater adhesion of Mussels by using DOPA-deficient recombinant Mussel Adhesive Proteins. Considering the instability of DOPA in an oxidative environment, the cation–π interaction in DOPA-deficient biopolymers provides a complementary cross-linking mechanism for the design of novel underwater Adhesives.
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glycosylated hydroxytryptophan in a Mussel Adhesive Protein from perna viridis
Journal of Biological Chemistry, 2009Co-Authors: Hua Zhao, Dong Soo Hwang, Jason Sagert, Herbert J WaiteAbstract:Abstract The 3,4-dihydroxyphenyl-l-alanine (Dopa)-containing Proteins of Mussel byssus play a critical role in wet adhesion and have inspired versatile new synthetic strategies for Adhesives and coatings. Apparently, however, not all Mussel Adhesive Proteins are beholden to Dopa chemistry. The cDNA-deduced sequence of Pvfp-1, a highly aromatic and redox active byssal coating Protein in the green Mussel Perna viridis, suggests that Dopa may be replaced by a post-translational modification of tryptophan. The N-terminal tryptophan-rich domain of Pvfp-1 contains 42 decapeptide repeats with the consensus sequences ATPKPW1TAW2K and APPPAW1TAW2K. A small collagen domain (18 Gly-X-Y repeats) is also present. Tandem mass spectrometry of isolated tryptic decapeptides has detected both C2-hexosylated tryptophan (W1) and C2-hexosylated hydroxytryptophan (W2), the latter of which is redox active. The UV absorbance spectrum of W2 is consistent with 7-hydroxytryptophan, which represents an intriguing new theme for bioinspired opportunistic wet adhesion.
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Recombinant Mussel Adhesive Protein as a gene delivery material.
Biotechnology and bioengineering, 2009Co-Authors: Dong Soo Hwang, Yoo Seong Choi, Kyoung Ro Kim, Seonghye Lim, Hyung Joon ChaAbstract:Efficient target gene delivery into eukaryotic cells is important for biotechnological research and gene therapy. Gene delivery based on Proteins, including histones, has recently emerged as a powerful non-viral DNA transfer technique. Here, we investigated the potential use of a recombinant Mussel Adhesive Protein, hybrid fp-151, as a gene delivery material, in view of its similar basic amino acid composition to histone Proteins, and cost-effective and high-level production in Escherichia coli. After confirming DNA binding affinity, we transfected mammalian cells (human 293T and mouse NIH/3T3) with foreign genes using hybrid fp-151 as the gene delivery carrier. Hybrid fp-151 displayed comparable transfection efficiency in both mammalian cell lines, compared to the widely used transfection agent, Lipofectamine 2000. Our results indicate that this Mussel Adhesive Protein may be used as a potential Protein-based gene-transfer mediator.
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Bulk Adhesive strength of recombinant hybrid Mussel Adhesive Protein.
Biofouling, 2009Co-Authors: Hyung Joon Cha, Seonghye Lim, Dong Soo Hwang, James D. White, Cristina R. Matos-pérez, Jonathan J. WilkerAbstract:Mussel Adhesive Proteins (MAPs) have received increased attention as potential biomedical and environmental friendly Adhesives. However, practical application of MAPs has been severely limited by u...
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expression of functional recombinant Mussel Adhesive Protein type 3a in escherichia coli
Biotechnology Progress, 2008Co-Authors: Dong Soo Hwang, Youngsoo Gim, Hyung Joon ChaAbstract: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.
Yoo Seong Choi - One of the best experts on this subject based on the ideXlab platform.
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RECOMBINANT Mussel Adhesive Protein AS A GENE DELIVERY MATERIAL
'Wiley', 2019Co-Authors: Ds Hwang, Yoo Seong Choi, Kyoung Ro Kim, Seonghye Lim, Cha, Hyung JoonAbstract:Efficient target gene delivery into eukaryotic cells is important for biotechnological research and gene therapy. Gene delivery based on Proteins, including histones, has recently emerged as a powerful non-viral DNA transfer technique. Here, we investigated the potential use of a recombinant Mussel Adhesive Protein, hybrid fp-151, as a gene delivery material, in view of its similar basic amino acid composition to histone Proteins, and cost-effective and high-level production in Escherichia coli. After confirming DNA binding affinity, we transfected mammalian cells (human 293T and mouse NIH/3T3) with foreign genes using hybrid fp-151 as the gene delivery carrier. Hybrid fp-151 displayed comparable transfection efficiency in both mammalian cell lines, compared to the widely used transfection agent, Lipofectamine (TM) 2000. Our results indicate that this Mussel Adhesive Protein may be used as a potential Protein-based gene-transfer mediator.Efficient target gene delivery into eukaryotic cells is important for biotechnological research and gene therapy. Gene delivery based on Proteins, including histones, has recently emerged as a powerful non-viral DNA transfer technique. Here, we investigated the potential use of a recombinant Mussel Adhesive Protein, hybrid fp-151, as a gene delivery material, in view of its similar basic amino acid composition to histone Proteins, and cost-effective and high-level production in Escherichia coli. After confirming DNA binding affinity, we transfected mammalian cells (human 293T and mouse NIH/3T3) with foreign genes using hybrid fp-151 as the gene delivery carrier. Hybrid fp-151 displayed comparable transfection efficiency in both mammalian cell lines, compared to the widely used transfection agent, Lipofectamine (TM) 2000. Our results indicate that this Mussel Adhesive Protein may be used as a potential Protein-based gene-transfer mediator.X1
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Mussel Adhesive Protein as an environmentally-friendly harmless wood furniture Adhesive
International Journal of Adhesion and Adhesives, 2016Co-Authors: Young Hoon Song, Yoo Seong Choi, Bong-hyuk ChoiAbstract:Abstract Recent Adhesive technologies have focused on the development of high-quality and eco-friendly Adhesives. Thus, there is a gradual shift from the currently used chemical-based Adhesives toward harmless Adhesives with improved quality and performance. Here, we evaluated the potential use of bacteria-produced recombinant Mussel Adhesive Protein (MAP) as a harmless wood furniture Adhesive. We formulated a MAP wood Adhesive as an inclusion body type for economical preparation, and we confirmed its harmlessness through the non-detection of volatile organic compounds and heavy metals. The formulated MAP showed sufficiently strong bulk Adhesive strength for the dried gluing of wood adherends. We also found that the formulated MAP wood Adhesive exhibits robust adhesion in various environmental conditions, including open assembly times, incubation times, temperatures, and humidity levels. In summary, the developed recombinant MAP could be successfully used as a promising environmentally-friendly, harmless wood furniture Adhesive.
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A comparative study on the bulk Adhesive strength of the recombinant Mussel Adhesive Protein fp-3
Biofouling, 2013Co-Authors: Byeongseon Yang, Yoo Seong Choi, Jeong Hyun Seo, Dong Gyun Kang, Hyung Joon ChaAbstract: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 bioadh...
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in vivo post translational modifications of recombinant Mussel Adhesive Protein in insect cells
Biotechnology Progress, 2011Co-Authors: Seonghye Lim, Yoo Seong Choi, Kyoung Ro Kim, Dae-kyum Kim, Daehee Hwang, Hyung Joon ChaAbstract: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.
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A Mussel Adhesive Protein Fused with the BC Domain of Protein A is a Functional Linker Material that Efficiently Immobilizes Antibodies onto Diverse Surfaces
Advanced Functional Materials, 2011Co-Authors: Chang Sup Kim, Yoo Seong Choi, Jeong Hyun Seo, Jieun Lee, Hyung Joon ChaAbstract:The efficient immobilization of antibodies onto solid surfaces is vital for the sensitivity and specificity of various immunoassays and immunosensors. A novel linker Protein, BC-MAP, is designed and produced in Escherichia coli by genetically fusing Mussel Adhesive Protein (MAP) with two domains (B and C) of Protein A (antibody-binding Protein) for efficient antibody immobilization on diverse surfaces. Through direct surface-coating analyses, it is found that BC-MAP successfully coats diverse surfaces including glass, polymers, and metals, but the BC domain alone does not. Importantly, antibodies are efficiently immobilized on BC-MAP-coated surfaces, and the immobilized antibodies interact selectively with their corresponding antigen. Quartz-crystal-microbalance analyses show that BC-MAP has excellent antibody-binding ability compared to that of BC Protein on gold surfaces. These results demonstrate that the MAP domain, with uniquely strong underwater Adhesive properties, plays a role in the direct and efficient coating of BC-MAP molecules onto diverse surfaces that lack additional surface treatment, and the BC domain of BC-MAP contributes to the selective and oriented immobilization of antibodies on BC-MAP-coated surfaces. Thus, the BC-MAP fusion Protein could be a valuable novel linker material for the facile and efficient immobilization of antibodies onto diverse solid supports.
Dongchuan Yang - One of the best experts on this subject based on the ideXlab platform.
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Mussel Adhesive Protein fused with ve cadherin extracellular domain promotes endothelial cell tight junctions and in vivo endothelization recovery of vascular stent
Journal of Biomedical Materials Research Part B, 2020Co-Authors: Dongchuan Yang, Yi Wang, Yuhua Huang, Tianhan Li, Nan Wang, Colm Durkan, Junli Huang, Guixue WangAbstract:: Improving the surface properties of vascular stents to accelerate endothelialization in vivo could play an important role in minimizing the risk of late thrombosis. We previously showed that Mussel Adhesive Protein fused with VE-cadherin extracellular domain (VE-M) specifically triggered endothelial cell adhesion in vitro. In this study, using stent implants coated with VE-M, we evaluated the clinical applicability of VE-M in endothelialization recovery in vivo. First, we explored the effect of VE-M on hemocompatibility and tight junctions between endothelial cells (ECs) in vitro. VE-M significantly inhibited platelet adhesion and promoted EC proliferation. Furthermore, VE-M drastically increased the centralization of F-actin in human umbilical vein endothelial cells (HUVECs) along the cell contacts, reduced fluorescein isothiocyanate (FITC)-dextran transport across the HUVECs, and elevated expression levels of tight junction Proteins (TJPs) in ECs. We then evaluated the effect of VE-M on endothelialization recovery in vivo through implantation of vascular stents. At 1 day after implantation, stents coated with VE-M recruited more endothelial progenitor cells (EPCs) than bare stents. At 7 days after implantation, VE-M stents had a greater coverage of ECs than bare stents. At 1 month after implantation, ECs on VE-M stents were appropriately elliptical in morphology and closely resembled physiological morphology. Hematoxylin-eosin (HE) staining revealed little in-stent neointima formation on VE-M stents, and SEM images revealed that smooth endothelium had formed on VE-M stents without adherent platelets. Taken together, these findings indicate that VE-M accelerates in vivo endothelialization of vascular stents via recruitment of EPCs and promotes endothelium formation and could be explored as a potential bioactive coating for vascular implant. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:94-103, 2020.
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Mussel Adhesive Protein fused with VE-cadherin extracellular domain promotes endothelial-cell tight junctions and in vivo endothelization recovery of vascular stent.
Journal of biomedical materials research. Part B Applied biomaterials, 2019Co-Authors: Dongchuan Yang, Yi Wang, Yuhua Huang, Nan Wang, Colm Durkan, Junli Huang, Wenhua Yan, Juhui Qiu, Tieying YinAbstract:Improving the surface properties of vascular stents to accelerate endothelialization in vivo could play an important role in minimizing the risk of late thrombosis. We previously showed that Mussel Adhesive Protein fused with VE-cadherin extracellular domain (VE-M) specifically triggered endothelial cell adhesion in vitro. In this study, using stent implants coated with VE-M, we evaluated the clinical applicability of VE-M in endothelialization recovery in vivo. First, we explored the effect of VE-M on hemocompatibility and tight junctions between endothelial cells (ECs) in vitro. VE-M significantly inhibited platelet adhesion and promoted EC proliferation. Furthermore, VE-M drastically increased the centralization of F-actin in human umbilical vein endothelial cells (HUVECs) along the cell contacts, reduced fluorescein isothiocyanate (FITC)-dextran transport across the HUVECs, and elevated expression levels of tight junction Proteins (TJPs) in ECs. We then evaluated the effect of VE-M on endothelialization recovery in vivo through implantation of vascular stents. At 1 day after implantation, stents coated with VE-M recruited more endothelial progenitor cells (EPCs) than bare stents. At 7 days after implantation, VE-M stents had a greater coverage of ECs than bare stents. At 1 month after implantation, ECs on VE-M stents were appropriately elliptical in morphology and closely resembled physiological morphology. Hematoxylin-eosin (HE) staining revealed little in-stent neointima formation on VE-M stents, and SEM images revealed that smooth endothelium had formed on VE-M stents without adherent platelets. Taken together, these findings indicate that VE-M accelerates in vivo endothelialization of vascular stents via recruitment of EPCs and promotes endothelium formation and could be explored as a potential bioactive coating for vascular implant. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:94-103, 2020.
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Mussel Adhesive Protein fused with ve cadherin domain specifically triggers endothelial cell adhesion
Journal of Materials Chemistry B, 2018Co-Authors: Dongchuan Yang, Junli Huang, Juhui Qiu, Yinping Zhao, Guixue WangAbstract:Endothelium is the only known completely non-thrombogenic material. In the present study, a strategy to mimic the Adhesive interactions of endothelial cells (ECs) to alter the vascular microenvironment was established and applied to directing the behaviour of cells. To facilitate the regeneration of a functional endothelium in vascular lesions, we designed a recombinant Mussel foot Protein (Mfp-5) fused with the VE-cadherin extracellular domain EC1-2, termed VE-M. Surface coating analysis showed that recombinant VE-M successfully formed a coating on substrate materials with uniform nanorods, low roughness, and sufficient hydrophilicity. We then evaluated the effects of VE-M on the adhesion of ECs and the capture of endothelial progenitor cells (EPCs). The result demonstrated that VE-M efficiently promoted the adhesion of ECs and EPCs. The number of ECs and EPCs on VE-M was 5.5- and 1.8-fold higher, respectively, than that on bare 316L SS under static conditions, whereas there was no significant difference in the number of captured smooth muscle cells (SMCs) between VE-M and other substrates. In addition, the number of EPCs captured by VE-M was approximately four times higher than that captured by 316L SS under dynamic conditions. In particular, the result of the neutralization test indicated that VE-M specifically triggered ECs’ adhesion via the interaction of VE-cadherin EC1-2. Further investigation showed that VE-M significantly increased the levels of endogenous VE-cadherin in HUVECs as well as the endothelial eNOS content, with little or no endothelial inflammation. Our results showed that VE-M could be a promising biomimetic modification for accelerating endothelialization and vascularization in tissue engineering.
