Publications by authors named "Jeong Hyun Seo"

40 Publications

Glycan chip based on structure-switchable DNA linker for on-chip biosynthesis of cancer-associated complex glycans.

Nat Commun 2021 03 2;12(1):1395. Epub 2021 Mar 2.

Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Republic of Korea.

On-chip glycan biosynthesis is an effective strategy for preparing useful complex glycan sources and for preparing glycan-involved applications simultaneously. However, current methods have some limitations when analyzing biosynthesized glycans and optimizing enzymatic reactions, which could result in undefined glycan structures on a surface, leading to unequal and unreliable results. In this work, a glycan chip is developed by introducing a pH-responsive i-motif DNA linker to control the immobilization and isolation of glycans on chip surfaces in a pH-dependent manner. On-chip enzymatic glycosylations are optimized for uniform biosynthesis of cancer-associated Globo H hexasaccharide and its related complex glycans through stepwise quantitative analyses of isolated products from the surface. Successful interaction analyses of the anti-Globo H antibody and MCF-7 breast cancer cells with on-chip biosynthesized Globo H-related glycans demonstrate the feasibility of the structure-switchable DNA linker-based glycan chip platform for on-chip complex glycan biosynthesis and glycan-involved applications.
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http://dx.doi.org/10.1038/s41467-021-21538-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925590PMC
March 2021

QTL Mapping and Candidate Gene Analysis for Pod Shattering Tolerance in Soybean ().

Plants (Basel) 2020 Sep 8;9(9). Epub 2020 Sep 8.

Department of Plant Bioscience, Pusan National University, Miryang 50463, Korea.

Pod shattering is an important reproductive process in many wild species. However, pod shattering at the maturing stage can result in severe yield loss. The objectives of this study were to discover quantitative trait loci (QTLs) for pod shattering using two recombinant inbred line (RIL) populations derived from an elite cultivar having pod shattering tolerance, namely "Daewonkong", and to predict novel candidate QTL/genes involved in pod shattering based on their allele patterns. We found several QTLs with more than 10% phenotypic variance explained (PVE) on seven different chromosomes and found a novel candidate QTL on chromosome 16 () from the allele patterns in the QTL region. Out of the 41 annotated genes in the QTL region, six were found to contain SNP (single-nucleotide polymorphism)/indel variations in the coding sequence of the parents compared to the soybean reference genome. Among the six potential candidate genes, , one of the genes with known function, showed a highly differential expression levels between the tolerant and susceptible parents in the growth stages R3 to R6. Further, is a homolog of in , whose function is related to abscisic acid catabolism. The results provide useful information to understand the genetic mechanism of pod shattering and could be used for improving the efficiency of marker-assisted selection for developing varieties of soybeans tolerant to pod shattering.
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http://dx.doi.org/10.3390/plants9091163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7569788PMC
September 2020

Electrohydrodynamic Sprayable Amphiphilic Polysaccharide-Clasped Nanoscale Self-Assembly for Imaging.

ACS Appl Mater Interfaces 2020 Sep 25;12(35):38899-38905. Epub 2020 Aug 25.

School of Chemical Engineering, Yeungnam University, 280 Daehakro, Gyeongsan, Gyeongbuk 38541, Korea.

The work presented in this report demonstrates that amphiphilic polysaccharide-clasped self-assembly (Amp-SA) with nanometer size, encapsulating hydrophobic nanoparticles (NPs) can be generated via electrohydrodynamic spraying. It is observed that the formation of hydrophobic NP-encapsulated Amp-SA is dependent on the surface chemistry of NPs. The citrate-coated magnetic NPs (MNPs-Cit) were also prepared and compared. The hydrophobic magnetic NP-encapsulated Amp-SA (Amp-SA-M) exhibited around 2.7-2.8-fold higher values in relaxivity than that of MNPs-Cit. In addition, the resulting Amp-SA-M achieved ∼17.2-fold higher values in / ratios than MNPs-Cit. The enhanced performances in magnetic transverse () relaxivity and / ratio as well as the behavior of Amp-SA-M suggest the potential of Amp-SA-M as a promising MRI nanoprobe. This approach based on the nature-originated amphiphilic biopolymers may provide a novel insight into electrohydrodynamic techniques that have the ability to create various nanostructures, encapsulating high-quality hydrophobic nanomaterials for applications in diverse biotechnology.
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http://dx.doi.org/10.1021/acsami.0c07473DOI Listing
September 2020

Polysaccharide-Hydrophobic Nanoparticle Hybrid Nanoclusters for Enhanced Performance in Magnetic Resonance/Photoacoustic Imaging.

Biomacromolecules 2019 11 9;20(11):4150-4157. Epub 2019 Oct 9.

School of Chemical Engineering , Yeungnam University , 280 Daehakro , Gyeongsan , Gyeongbuk 38541 , Korea.

Polysaccharide-nanoparticle (NP) hybrid nanoclusters have great potential to revitalize diverse bioapplications; however, fabricating polysaccharide-based hybrid nanoclusters composed of high-quality NPs generated in the organic phase remains a challenge. Here, using calcium alginate as a polysaccharide/tetramethylammonium hydroxide (TMAOH) combination, we report a novel approach to the design of alginate-hydrophobic magnetic-plasmonic core-shell (MPCS) NP hybrid nanoclusters (A-MPCS HNCs). Furthermore, we observe the dependence of the formation of A-MPCS HNCs on the TMAOH concentration. The enhanced performance in both magnetic resonance relaxivity and photoacoustic (PA) signals and the biocompatibility/bioactivity as well as the in vivo performance of A-MPCS HNCs shows them to be a promising magnetic resonance/photoacoustic dual-mode imaging agent. Our strategy could open doors to the use of other precious high-quality nanomaterials created in the organic phase via well-established synthetic chemistry in the design of alginate-hydrophobic nanomaterial hybrid nanoclusters, giving rise to novel and multifarious bioapplications.
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http://dx.doi.org/10.1021/acs.biomac.9b01008DOI Listing
November 2019

On-chip biosynthesis of GM1 pentasaccharide-related complex glycans.

Chem Commun (Camb) 2018 Dec;55(1):71-74

School of Chemistry and Biochemistry, Yeungnam University, Gyeongsan 38541, Korea.

A functional glycan chip combined with on-chip enzymatic glycosylation was developed to prepare complex glycan sources and to apply glycan-involved applications simultaneously. GM3 trisaccharide, GM2 tetrasaccharide, and GM1 pentasaccharide were successfully directly biosynthesized on lactose-immobilized surfaces through three consecutive glycosyltransferase reactions along with small amounts of enzymes and donors, without any additional processes. Biosynthesized GM1 pentasaccharide-related complex glycans were demonstrated to provide information on the substrate specificity of whole cholera toxin. Thus, the proposed on-chip glycan biosynthesis system can provide a new direction toward obtaining complex glycan sources and complex glycan-involved applications such as glycan-protein interaction analysis and glycan biomarker-based diagnosis.
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http://dx.doi.org/10.1039/c8cc06526hDOI Listing
December 2018

Surface Design of Eu-Doped Iron Oxide Nanoparticles for Tuning the Magnetic Relaxivity.

ACS Appl Mater Interfaces 2018 Aug 20;10(30):25080-25089. Epub 2018 Jul 20.

School of Chemical Engineering , Yeungnam University , 280 Daehakro , Gyeongsan , Gyeongbuk 38541 , Korea.

Relaxivity tuning of nanomaterials with the intrinsic T- T dual-contrast ability has great potential for MRI applications. Until now, the relaxivity tuning of T and T dual-modal MRI nanoprobes has been accomplished through the dopant, size, and morphology of the nanoprobes, leaving room for bioapplications. However, a surface engineering method for the relaxivity tuning was seldom reported. Here, we report the novel relaxivity tuning method based on the surface engineering of dual-mode T- T MRI nanoprobes (DMNPs), along with protein interaction monitoring with the DMNPs as a potential biosensor application. Core nanoparticles (NPs) of europium-doped iron oxide (EuIO) are prepared by a thermal decomposition method. As surface materials, citrate (Cit), alendronate (Ale), and poly(maleic anhydride- alt-1-octadecene)/poly(ethylene glycol) (PP) are employed for the relaxivity tuning of the NPs based on surface engineering, resulting in EuIO-Cit, EuIO-Ale, and EuIO-PP, respectively. The key achievement of the current study is that the surface materials of the DMNP have significant impacts on the r and r relaxivities. The correlation between the hydrophobicity of the surface material and longitudinal relaxivity ( r) of EuIO NPs presents an exponential decay feature. The r relaxivity of EuIO-Cit is 13.2-fold higher than that of EuIO-PP. EuIO can act as T- T dual-modal (EuIO-Cit) or T-dominated MRI contrast agents (EuIO-PP) depending on the surface engineering. The feasibility of using the resulting nanosystem as a sensor for environmental changes, such as albumin interaction, was also explored. The albumin interaction on the DMNP shows both T and T relaxation time changes as mutually confirmative information. The relaxivity tuning approach based on the surface engineering may provide an insightful strategy for bioapplications of DMNPs and give a fresh impetus for the development of novel stimuli-responsive MRI nanoplatforms with T and T dual-modality for various biomedical applications.
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http://dx.doi.org/10.1021/acsami.8b06569DOI Listing
August 2018

Survival of Verwey transition in gadolinium-doped ultrasmall magnetite nanoparticles.

Nanoscale 2017 Sep;9(37):13976-13982

Korea Multi-purpose Accelerator Complex, Korea Atomic Energy Research Institute, Gyeongju, 305-353, Korea.

We have demonstrated that the Verwey transition, which is highly sensitive to impurities, survives in anisotropic Gd-doped magnetite nanoparticles. Transmission electron microscopy analysis shows that the nanoparticles are uniformly distributed. X-ray photoelectron spectroscopy and EDS mapping analysis confirm Gd-doping on the nanoparticles. The Verwey transition of the Gd-doped magnetite nanoparticles is robust and the temperature dependence of the magnetic moment (zero field cooling and field cooling) shows the same behaviour as that of the Verwey transition in bulk magnetite, at a lower transition temperature (∼110 K). In addition, irregularly shaped nanoparticles do not show the Verwey transition whereas square-shaped nanoparticles show the transition. Mössbauer spectral analysis shows that the slope of the magnetic hyperfine field and the electric quadrupole splitting change at the same temperature, meaning that the Verwey transition occurs at ∼110 K. These results would provide new insights into understanding the Verwey transition in nano-sized materials.
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http://dx.doi.org/10.1039/c7nr03684aDOI Listing
September 2017

Selection of affinity peptides for interference-free detection of cholera toxin.

Biosens Bioelectron 2018 Jan 31;99:289-295. Epub 2017 Jul 31.

Department of Pharmaceutical Engineering, Daegu Haany University, Gyeongsan 38610, Republic of Korea. Electronic address:

Cholera toxin is a major virulent agent of Vibrio cholerae, and it can rapidly lead to severe dehydration, shock, causing death within hours without appropriate clinical treatments. In this study, we present a method wherein unique and short peptides that bind to cholera toxin subunit B (CTX-B) were selected through M13 phage display. Biopanning over recombinant CTX-B led to rapid screening of a unique peptide with an amino acid sequence of VQCRLGPPWCAK, and the phage-displayed peptides analyzed using ELISA, were found to show specific affinities towards CTX-B. To address the use of affinity peptides in development of the biosensor, sequences of newly selected peptides were modified and chemically synthesized to create a series of affinity peptides. Performance of the biosensor was studied using plasmonic-based optical techniques: localized surface plasmon resonance (LSPR) and surface-enhanced Raman scattering (SERS). The limit of detection (LOD) obtained by LSPR with 3σ-rule was 1.89ng/mL, while SERS had a LOD of 3.51pg/mL. In both cases, the sensitivity was much higher than the previously reported values, and our sensor system was specific towards actual CTX-B secreted from V. cholera, but not for CTX-AB.
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http://dx.doi.org/10.1016/j.bios.2017.07.075DOI Listing
January 2018

Versatile signal peptide of Flavobacterium-originated organophosphorus hydrolase for efficient periplasmic translocation of heterologous proteins in Escherichia coli.

Biotechnol Prog 2016 07 27;32(4):848-54. Epub 2016 Apr 27.

Dept. of Chemical Engineering, Pohang University of Science and Technology, Pohang, 790-784, Korea.

Organophosphorus hydrolase (OPH) from Flavobacterium species is a membrane-associated homodimeric metalloenzyme and has its own signal peptide in its N-terminus. We found that OPH was translocated into the periplasmic space when the original signal peptide-containing OPH was expressed in recombinant Escherichia coli even though its translocation efficiency was relatively low. To investigate the usability of this OPH signal peptide for periplasmic expression of heterologous proteins in an E. coli system, we employed green fluorescent protein (GFP) as a cytoplasmic folding reporter and alkaline phosphatase (ALP) as a periplasmic folding reporter. We found that the OPH signal peptide was able to use both twin-arginine translocation (Tat) and general secretory (Sec) machineries by switching translocation pathways according to the nature of target proteins in E. coli. These results might be due to the lack of Sec-avoidance sequence in the c-region and a moderate hydrophobicity of the OPH signal peptide. Interestingly, the OPH signal peptide considerably enhanced the translocation efficiencies for both GFP and ALP compared with commonly used TorA and PelB signal peptides that have Tat and Sec pathway dependences, respectively. Therefore, this OPH signal peptide could be successfully used in recombinant E. coli system for efficient periplasmic production of target protein regardless of the subcellular localization where functional folding of the protein occurs. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:848-854, 2016.
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http://dx.doi.org/10.1002/btpr.2274DOI Listing
July 2016

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

BMC Biotechnol 2016 Feb 16;16:16. Epub 2016 Feb 16.

School of Chemical Engineering, Yeungnam University, Gyeongsan, 712-749, Korea.

Background: von 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.

Results: Here, 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.

Conclusions: This 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.
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http://dx.doi.org/10.1186/s12896-016-0247-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4754843PMC
February 2016

Hybrid microarray based on double biomolecular markers of DNA and carbohydrate for simultaneous genotypic and phenotypic detection of cholera toxin-producing Vibrio cholerae.

Biosens Bioelectron 2016 May 21;79:398-405. Epub 2015 Dec 21.

Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea. Electronic address:

Life-threatening diarrheal cholera is usually caused by water or food contaminated with cholera toxin-producing Vibrio cholerae. For the prevention and surveillance of cholera, it is crucial to rapidly and precisely detect and identify the etiological causes, such as V. cholerae and/or its toxin. In the present work, we propose the use of a hybrid double biomolecular marker (DBM) microarray containing 16S rRNA-based DNA capture probe to genotypically identify V. cholerae and GM1 pentasaccharide capture probe to phenotypically detect cholera toxin. We employed a simple sample preparation method to directly obtain genomic DNA and secreted cholera toxin as target materials from bacterial cells. By utilizing the constructed DBM microarray and prepared samples, V. cholerae and cholera toxin were detected successfully, selectively, and simultaneously; the DBM microarray was able to analyze the pathogenicity of the identified V. cholerae regardless of whether the bacteria produces toxin. Therefore, our proposed DBM microarray is a new effective platform for identifying bacteria and analyzing bacterial pathogenicity simultaneously.
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http://dx.doi.org/10.1016/j.bios.2015.12.073DOI Listing
May 2016

Surface-independent antibacterial coating using silver nanoparticle-generating engineered mussel glue.

ACS Appl Mater Interfaces 2014 Nov 23;6(22):20242-53. Epub 2014 Oct 23.

Department of Chemical Engineering, Pohang University of Science and Technology , Pohang 790-784, Korea.

During implant surgeries, antibacterial agents are needed to prevent bacterial infections, which can cause the formation of biofilms between implanted materials and tissue. Mussel adhesive proteins (MAPs) derived from marine mussels are bioadhesives that show strong adhesion and coating ability on various surfaces even in wet environment. Here, we proposed a novel surface-independent antibacterial coating strategy based on the fusion of MAP to a silver-binding peptide, which can synthesize silver nanoparticles having broad antibacterial activity. This sticky recombinant fusion protein enabled the efficient coating on target surface and the easy generation of silver nanoparticles on the coated-surface under mild condition. The biosynthesized silver nanoparticles showed excellent antibacterial efficacy against both Gram-positive and Gram-negative bacteria and also revealed good cytocompatibility with mammalian cells. In this coating strategy, MAP-silver binding peptide fusion proteins provide hybrid environment incorporating inorganic silver nanoparticle and simultaneously mediate the interaction of silver nanoparticle with surroundings. Moreover, the silver nanoparticles were fully synthesized on various surfaces including metal, plastic, and glass by a simple, surface-independent coating manner, and they were also successfully synthesized on a nanofiber surface fabricated by electrospinning of the fusion protein. Thus, this facile surface-independent silver nanoparticle-generating antibacterial coating has great potential to be used for the prevention of bacterial infection in diverse biomedical fields.
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http://dx.doi.org/10.1021/am505784kDOI Listing
November 2014

Engineered whole-cell biocatalyst-based detoxification and detection of neurotoxic organophosphate compounds.

Biotechnol Adv 2014 May-Jun;32(3):652-62. Epub 2014 Apr 26.

Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea. Electronic address:

The development of efficient tools is required for the eco-friendly detoxification and effective detection of neurotoxic organophosphates (OPs). Although enzymes have received significant attention as biocatalysts because of their high specific activity, the uneconomic and labor-intensive processes of enzyme production and purification make their broad use in practical applications difficult. Because whole-cell systems offer several advantages compared with free enzymes, including high stability, a reduced purification requirement, and low preparation cost, they have been suggested as promising biocatalysts for the detoxification and detection of OPs. To develop efficient whole-cell biocatalysts with enhanced activity and a broad spectrum of substrate specificity, several factors have been considered, namely the selected strains, the chosen OP-hydrolyzing enzymes, where enzymes are localized in a cell, and which enhancer will assist the expression, function, and folding of the enzyme. In this article, we review the current investigative progress in the development of engineered whole-cell biocatalysts with excellent OP-hydrolyzing activity, a broad spectrum of substrate specificity, and outstanding stability for the detoxification and detection of OPs.
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http://dx.doi.org/10.1016/j.biotechadv.2014.04.010DOI Listing
January 2015

Highly purified mussel adhesive protein to secure biosafety for in vivo applications.

Microb Cell Fact 2014 Apr 11;13(1):52. Epub 2014 Apr 11.

Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea.

Background: Unique adhesive and biocompatibility properties of mussel adhesive proteins (MAPs) are known for their great potential in many tissue engineering and biomedical applications. Previously, it was successfully demonstrated that redesigned hybrid type MAP, fp-151, mass-produced in Gram-negative bacterium Escherichia coli, could be utilized as a promising adhesive biomaterial. However, purification of recombinant fp-151 has been unsatisfactory due to its adhesive nature and polarity which make separation of contaminants (especially, lipopolysaccharide, a toxic Gram-negative cell membrane component) very difficult.

Results: In the present work, we devised a high resolution purification approach to secure safety standards of recombinant fp-151 for the successful use in in vivo applications. Undesirable impurities were remarkably eliminated as going through sequential steps including treatment with multivalent ion and chelating agent for cell membrane washing, mechanical cell disruption, non-ionic surfactant treatment for isolated inclusion body washing, acid extraction of washed inclusion body, and ion exchange chromatography purification of acid extracted sample. Through various analyses, such as high performance liquid chromatographic purity assay, limulus amoebocyte lysate endotoxin assay, and in vitro mouse macrophage cell tests on inflammation, viability, cytotoxicity, and apoptosis, we confirmed the biological safety of bacterial-derived purified recombinant fp-151.

Conclusions: Through this purification design, recombinant fp-151 achieved 99.90% protein purity and 99.91% endotoxin reduction that nearly no inflammation response was observed in in vitro experiments. Thus, the highly purified recombinant MAP would be successfully used as a safety-secured in vivo bioadhesive for tissue engineering and biomedical applications.
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http://dx.doi.org/10.1186/1475-2859-13-52DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3989845PMC
April 2014

Mussel-mimetic protein-based adhesive hydrogel.

Biomacromolecules 2014 May 8;15(5):1579-85. Epub 2014 Apr 8.

School of Interdisciplinary Bioscience and Bioengineering, ‡Ocean Science and Technology Institute, §School of Environmental Science and Engineering, ∥Department of Chemical Engineering, and ⊥Integrative Biosciences and Biotechnology, Pohang University of Science and Technology , Pohang 790-784, Korea.

Hydrogel systems based on cross-linked polymeric materials which could provide both adhesion and cohesion in wet environment have been considered as a promising formulation of tissue adhesives. Inspired by marine mussel adhesion, many researchers have tried to exploit the 3,4-dihydroxyphenylalanine (DOPA) molecule as a cross-linking mediator of synthetic polymer-based hydrogels which is known to be able to achieve cohesive hardening as well as adhesive bonding with diverse surfaces. Beside DOPA residue, composition of other amino acid residues and structure of mussel adhesive proteins (MAPs) have also been considered important elements for mussel adhesion. Herein, we represent a novel protein-based hydrogel system using DOPA-containing recombinant MAP. Gelation can be achieved using both oxdiation-induced DOPA quinone-mediated covalent and Fe(3+)-mediated coordinative noncovalent cross-linking. Fe(3+)-mediated hydrogels show deformable and self-healing viscoelastic behavior in rheological analysis, which is also well-reflected in bulk adhesion strength measurement. Quinone-mediated hydrogel has higher cohesive strength and can provide sufficient gelation time for easier handling. Collectively, our newly developed MAP hydrogel can potentially be used as tissue adhesive and sealant for future applications.
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http://dx.doi.org/10.1021/bm4017308DOI Listing
May 2014

Multifunctional adhesive silk fibroin with blending of RGD-bioconjugated mussel adhesive protein.

Biomacromolecules 2014 Apr 18;15(4):1390-8. Epub 2014 Mar 18.

Department of Chemical Engineering, Pohang University of Science and Technology , Pohang 790-784, Korea.

Silk has recently been exploited in various fields due to its superior mechanical properties. However, this material's lack of biological functions and relatively poor biodegradation have hindered its wide use in applications related to cells and tissues. Here, we improved the overall characteristics of silkworm silk fibroin (SF) by introduction of RGD peptide-fused recombinant mussel adhesive protein (MAP-RGD). Simple blending of MAP-RGD provided not only bulk-scale adhesive ability but also microscale adhesiveness to cells and various biomolecules. MAP-RGD-blended SF fibers supported enhanced adhesion, proliferation, and spreading of mammalian cells as well as the efficient attachment of biomolecules, including carbohydrate and protein. In addition, the hydrophilicity, swelling, and biodegradability of the MAP-RGD-blended SF material were improved without notable hampering of the original mechanical properties of SF. Therefore, the adhesive silk fibrous scaffold could be successfully used in diverse biomedical engineering applications.
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http://dx.doi.org/10.1021/bm500001nDOI Listing
April 2014

Interfacial tension of complex coacervated mussel adhesive protein according to the Hofmeister series.

Langmuir 2014 Feb 22;30(4):1108-15. Epub 2014 Jan 22.

Department of Chemical Engineering, Pohang University of Science and Technology , Pohang 790-784, Korea.

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 were found to follow the Hofmeister ordering. The tendency of interfacial tension between the coacervate and dilute phases in the presence of different types of Hofmeister salt ions could provide a better understanding of Hofmeister effects on complex coacervated materials based on the protein-polysaccharide system. This information can also be utilized for microencapsulation and adsorption by controlling intramolecular interactions. In addition, the injection molding dynamics of mussel byssus formation was potentially explained based on the measured interfacial tension of coacervated MAP.
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http://dx.doi.org/10.1021/la403680zDOI Listing
February 2014

Specific discrimination of three pathogenic Salmonella enterica subsp. enterica serotypes by carB-based oligonucleotide microarray.

Appl Environ Microbiol 2014 Jan 1;80(1):366-73. Epub 2013 Nov 1.

Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, South Korea.

It is important to rapidly and selectively detect and analyze pathogenic Salmonella enterica subsp. enterica in contaminated food to reduce the morbidity and mortality of Salmonella infection and to guarantee food safety. In the present work, we developed an oligonucleotide microarray containing duplicate specific capture probes based on the carB gene, which encodes the carbamoyl phosphate synthetase large subunit, as a competent biomarker evaluated by genetic analysis to selectively and efficiently detect and discriminate three S. enterica subsp. enterica serotypes: Choleraesuis, Enteritidis, and Typhimurium. Using the developed microarray system, three serotype targets were successfully analyzed in a range as low as 1.6 to 3.1 nM and were specifically discriminated from each other without nonspecific signals. In addition, the constructed microarray did not have cross-reactivity with other common pathogenic bacteria and even enabled the clear discrimination of the target Salmonella serotype from a bacterial mixture. Therefore, these results demonstrated that our novel carB-based oligonucleotide microarray can be used as an effective and specific detection system for S. enterica subsp. enterica serotypes.
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http://dx.doi.org/10.1128/AEM.02978-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911022PMC
January 2014

Structural evaluation of GM1-related carbohydrate-cholera toxin interactions through surface plasmon resonance kinetic analysis.

Analyst 2013 Nov;138(22):6924-9

Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea.

Surface plasmon resonance (SPR) can provide kinetic information about an interaction, and it can also be used to rapidly monitor dynamic processes, such as adsorption and degradation, without the need for sample labeling. Here, we employed SPR to analyze carbohydrate-protein interactions, particularly GM1-related carbohydrate-Vibrio cholera toxin interactions. The interaction between cholera toxin subunits A (ctxA) and B (ctxB) was similar to general ligand-receptor interactions. After the direct immobilization of thiol-containing GM1 pentasaccharide on a gold surface, the GM1-ctxB interaction kinetics were evaluated, and they showed a similar degree of kinetics as reported in previous reports. We found that ctxA had a high affinity for the GM1-ctxAB complex, although its equilibrium dissociation constant was 10 times lower than that of GM1-ctxB binding. Comparative analyses of GM1-related carbohydrate-ctxAB interactions were also conducted to determine the kinetic values of several GM1 analogues with different structures, although their kinetic values were one order of magnitude lower than those of the GM1-ctxAB interaction. The kinetic analysis results for the interactions of GM1 analogues and ctxAB indicated that the sialic acid thumb is important for recognition, and the terminal galactose and N-acetylgalactosamine fingers are required to stabilize the GM1-ctxAB interaction. Taken together, our results indicate that the direct immobilization of carbohydrate in an SPR-based analytical system can be used to evaluate the structural contribution of carbohydrate moieties in carbohydrate-protein interactions, as well as provide valuable information that can be used to understand the interactions.
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http://dx.doi.org/10.1039/c3an01312jDOI Listing
November 2013

Engineered Escherichia coli with periplasmic carbonic anhydrase as a biocatalyst for CO2 sequestration.

Appl Environ Microbiol 2013 Nov 23;79(21):6697-705. Epub 2013 Aug 23.

School of Interdisciplinary Bioscience and Bioengineering.

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.
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http://dx.doi.org/10.1128/AEM.02400-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811487PMC
November 2013

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

Biofouling 2013 13;29(5):483-90. Epub 2013 May 13.

Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Korea.

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.
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http://dx.doi.org/10.1080/08927014.2013.782541DOI Listing
November 2013

Biological removal of phosphate at low concentrations using recombinant Escherichia coli expressing phosphate-binding protein in periplasmic space.

Appl Biochem Biotechnol 2013 Nov 16;171(5):1170-7. Epub 2013 Mar 16.

Department of Biological and Environmental Engineering, Semyung University, Jecheon, 390-711, Korea,

During wastewater treatment, phosphate removal is an important and challenging process; thus, diverse technologies, including those derived from biological means, have been devised for efficient phosphate removal. Although conventional biological methods are effective in decreasing wastewater phosphate levels to ~1 mg/L, long periods of microbial adaptation are required for effective phosphate removal, and the removal efficiency of these methods is relatively poor at lower phosphate concentrations. In the present work, we constructed a recombinant Escherichia coli with periplasmic-expressed phosphate-binding protein (PBP) and investigated its biological removal ability for low phosphate levels. We found that the PBP-expressing recombinant E. coli cells showed efficient (> 94 %) removal of phosphate at low concentrations (0.2-1.0 mg/L) in a treated cell mass-dependent manner. Collectively, we propose that our PBP-expressing recombinant whole-cell system could be successfully used during wastewater treatment for the biological removal of low concentrations of phosphate.
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http://dx.doi.org/10.1007/s12010-013-0187-1DOI Listing
November 2013

Mussel adhesive protein-based whole cell array biosensor for detection of organophosphorus compounds.

Biosens Bioelectron 2013 Mar 17;41:199-204. Epub 2012 Aug 17.

Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea.

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 μg/cm(2)) and cell loading (4 OD(600)), detected paraoxon levels as low as 5 μM with high reproducibility, and its quantitative detection range was ~5-320 μM. 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.
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http://dx.doi.org/10.1016/j.bios.2012.08.022DOI Listing
March 2013

Functional interaction analysis of GM1-related carbohydrates and Vibrio cholerae toxins using carbohydrate microarray.

Anal Chem 2012 Aug 17;84(15):6884-90. Epub 2012 Jul 17.

Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea.

The development of analytical tools is important for understanding the infection mechanisms of pathogenic bacteria or viruses. In the present work, a functional carbohydrate microarray combined with a fluorescence immunoassay was developed to analyze the interactions of Vibrio cholerae toxin (ctx) proteins and GM1-related carbohydrates. Ctx proteins were loaded onto the surface-immobilized GM1 pentasaccharide and six related carbohydrates, and their binding affinities were detected immunologically. The analysis of the ctx-carbohydrate interactions revealed that the intrinsic selectivity of ctx was GM1 pentasaccharide ≫ GM2 tetrasaccharide > asialo GM1 tetrasaccharide ≥ GM3trisaccharide, indicating that a two-finger grip formation and the terminal monosaccharides play important roles in the ctx-GM1 interaction. In addition, whole cholera toxin (ctxAB(5)) had a stricter substrate specificity and a stronger binding affinity than only the cholera toxin B subunit (ctxB). On the basis of the quantitative analysis, the carbohydrate microarray showed the sensitivity of detection of the ctxAB(5)-GM1 interaction with a limit-of-detection (LOD) of 2 ng mL(-1) (23 pM), which is comparable to other reported high sensitivity assay tools. In addition, the carbohydrate microarray successfully detected the actual toxin directly secreted from V. cholerae, without showing cross-reactivity to other bacteria. Collectively, these results demonstrate that the functional carbohydrate microarray is suitable for analyzing toxin protein-carbohydrate interactions and can be applied as a biosensor for toxin detection.
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http://dx.doi.org/10.1021/ac301511tDOI Listing
August 2012

Attachment of hydrogel microstructures and proteins to glass via thiol-terminated silanes.

Colloids Surf B Biointerfaces 2012 Oct 27;98:1-6. Epub 2012 Apr 27.

Department of Biomedical Engineering, University of California, Davis, CA 95616, United States.

Micropatterning strategies often call for attachment of non-fouling biomaterials and immobilization of proteins in order to create biosensing surfaces or to control cell-surface interactions. Our laboratory has made frequent use of hydrogel photolithography - a micropatterning process for immobilizing poly(ethylene glycol) (PEG) hydrogel microstructures on glass surfaces. In the present study we explored the use of thiolsilane as a coupling layer for both covalent anchoring of hydrogel microstructures and covalent immobilization of proteins on glass. These new surfaces were compared to acryl-silane functionalized glass slides that allowed covalent attachment of gels but only physical adsorption of proteins as well as surfaces containing a mixture of both functional groups. We observed comparable attachment and retention of hydrogel microstructures on acryl and thiol-terminated silanes. Ellipsometry studies revealed presence of significantly higher level of proteins on thiol-functionalized glass. Overall, our studies demonstrate that thiol-silane functionalized glass surfaces may be used to create complex micropatterned surfaces comprised of covalently attached hydrogels and proteins. This simple and effective surface modification strategy will be broadly applicable in cellular engineering and biosensing studies employing hydrogel micropatterns.
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http://dx.doi.org/10.1016/j.colsurfb.2012.03.025DOI Listing
October 2012

Coexpression of molecular chaperone enhances activity and export of organophosphorus hydrolase in Escherichia coli.

Biotechnol Prog 2012 Jul 8;28(4):925-30. Epub 2012 Jun 8.

Dept. of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea.

Periplasmic secretion has been used in attempts to construct an efficient whole-cell biocatalyst with greatly reduced diffusion limitations. Previously, we developed recombinant Escherichia coli that express organophosphorus hydrolase (OPH) in the periplasmic space using the twin-arginine translocation (Tat) pathway to degrade environmental toxic organophosphate compounds. This system has the advantage of secreting protein into the periplasm after folding in the cytoplasm. However, when OPH was expressed with a Tat signal sequence in E. coli, we found that the predominant OPH was an insoluble premature form in the cytoplasm, and thus, the whole-cell OPH activity was significantly lower than its cell lysate activity. In this work, we, for the first time, used a molecular chaperone coexpression strategy to enhance whole-cell OPH activity by improving the periplasmic translocation of soluble OPH. We found that the effect of GroEL-GroES (GroEL/ES) assistance on the periplasmic localization of OPH was secretory pathway dependent. We observed a significant increase in the amount of soluble mature OPH when cytoplasmic GroEL/ES was expressed; this increase in the amount of mature OPH might be due to enhanced OPH folding in the cytoplasm. Importantly, the whole-cell OPH activity of the chaperone-coexpressing cells was ∼5.5-fold greater at 12 h after induction than that of cells that did not express the chaperone as a result of significant Tat-based periplasmic translocation of OPH in the chaperone-coexpressing cells. Collectively, these data suggest that molecular chaperones significantly enhance the whole-cell activity of periplasmic OPH-secreting cells, yielding an effective whole-cell biocatalyst system with highly reduced diffusion limitations.
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http://dx.doi.org/10.1002/btpr.1556DOI Listing
July 2012

Characterization of the GM1 pentasaccharide-Vibrio cholera toxin interaction using a carbohydrate-based electrochemical system.

Analyst 2012 Jun 10;137(12):2860-5. Epub 2012 May 10.

Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea.

Antibody- or DNA-based electrochemical systems have been developed widely for several decades, while carbohydrate-based electrochemical systems have been rarely reported. Herein, we used an electrochemical detection system to understand the molecular relationships in carbohydrate-protein interactions that can provide useful information about biological processes in living organisms. This system was also helpful for the development of potent biomedical agents. Electrochemical detection was achieved through the observation of electrochemical response changes of ferrocyanide solution that resulted from the interaction of carbohydrate and protein using a modified GM1 pentasaccharide containing an anchoring thiol group that was directly immobilized on a gold electrode. As the concentration of the GM1 pentasaccharide increased, the current decreased gradually and saturated after 2 nM. We also found that the drop in current depended on the size of the carbohydrate (larger size of the carbohydrate denoted a higher slope of the current reduction), indicating that the current could be modulated by the molecular size of the carbohydrate as well as its concentration. This system was able to detect very low concentrations of carbohydrate (down to 20 fM), which highlighted the advantage of the electrochemical system. Interestingly, we found that a potential shift at the maximum current occurred upon interaction with cholera toxin proteins. By comparing results for different sizes of GM1 analogues, we surmise that the potential shift is closely associated with the specificity for the carbohydrate-protein interaction. Collectively, a carbohydrate-based electrochemical system can be leveraged for the facile and rapid analysis of carbohydrate-protein interactions.
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http://dx.doi.org/10.1039/c2an16221kDOI Listing
June 2012

Specific multiplex analysis of pathogens using a direct 16S rRNA hybridization in microarray system.

Anal Chem 2012 Jun 11;84(11):4873-9. Epub 2012 May 11.

Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea.

For the rapid multiplex analysis of pathogens, 16S rRNAs from cell lysates were directly applied onto a DNA microarray at room temperature (RT) for RNA-DNA hybridization. To eliminate the labeling step, seven fluorescent-labeled detector probes were cohybridized with 16S rRNA targets and adjacent specific capture probes. We found that eight pathogens were successfully discriminated by the 16S rRNA-based direct method, which showed greater specificity than the polymerase chain reaction (PCR)-labeled method due to chaperone and distance effects. A new specificity criterion for a perfect match between RNA and DNA was suggested to be 21-41% dissimilarity using correlation analysis between the mismatch and the sequence according to the guanine-cytosine (GC) percentage or the distribution of mismatches. Six categories of food matrix (egg, meat, milk, rice, vegetable, and mixed) were also tested, and the target pathogen was successfully discriminated within statistically significant levels. Finally, we found that the intrinsic abundance of 16S rRNA molecules successfully substituted PCR-based amplification with a low limit of detection of 10-10(3) cells mL(-1) and a high quantitative linear correlation. Collectively, our suggested 16S rRNA-based direct method enables the highly sensitive, specific, and quantitative analysis of selected pathogens at RT within 2 h, even in food samples.
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http://dx.doi.org/10.1021/ac300476kDOI Listing
June 2012

Photolabile micropatterned surfaces for cell capture and release.

Chem Commun (Camb) 2011 Nov 5;47(43):11942-4. Epub 2011 Oct 5.

Department of Biomedical Engineering, University of California, Davis, 451 East Health Sciences Dr #2619, Davis, CA 95616, USA.

A method for capture and release of cells was developed using a photolabile linker and antibody-attached glass surface with a poly(ethylene glycol) (PEG)-pattern.
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http://dx.doi.org/10.1039/c1cc15046dDOI Listing
November 2011

Quantitative label-free characterization of avidin-biotin assemblies on silanized glass.

Anal Chem 2011 Sep 29;83(18):7173-8. Epub 2011 Aug 29.

Department of Chemistry, Texas A&M University, College Station, Texas, United States.

In this study, a time-of-flight secondary ion mass spectrometer TOF-SIMS, operating in the event-by-event bombardment/detection mode was used to characterize avidin-biotin assemblies on silane-modified glass substrates. SIMS was used to analyze several variants of the biointerface, including avidin physically adsorbed on a monofunctional acryl silane surface and covalently attached on monofunctional (amine terminated) and bifunctional (amine and acryl terminated) silanes. The goal of these studies was to determine density of avidin and biotin layers chemically or physically adsorbed on silanized glass substrate. An individual impact of a C(60) projectile used in this study creates a hemispherical crater (∼10 nm in diameter) and emits large numbers of secondary ions from the same nanovolume. Thus, a single impact enables one to unfold distinct secondary ions that span the thickness of the assembled film. This method was used to monitor the presence of glass, silane, and protein ions and to estimate the thickness and density of the avidin layer. In addition, we employed the double coincidence mass spectrometry approach to identify ions coemitted from a specific stratum of the biointerface. This approach was used to determine density of biotin and avidin immobilization while eliminating interferences from isobaric ions that originated from other constituents on the surface. Overall, novel TOF-SIMS quantitative approaches employed here were useful for examining complex biointerfaces and determining both lateral and in depth composition of the film.
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http://dx.doi.org/10.1021/ac2016085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3186069PMC
September 2011