52. Ae Sol Lee, Ram Hari Pokhrel, Hye-Seon Jeong, Manoj Sharma, Chang-Hyung Choi*, Jae-Hoon Chang*, and Chang Sup Kim*, Antibody-Responsive Triple-Layered Microcapsules Enable Spatially Programmed Sequential Chemoimmunotherapy, Under Review
Publications (+First author; *Corresponding author)
51. Jiwoo Shin+, Sanghoon Jung+, Ae Sol Lee*, and Chang Sup Kim*, Protein nanocages: Programmable biomaterial platforms for therapeutics, bioimaging, and diagnostics, Acta Biomater. Under Revision
50. Min Ki Lee and Chang Sup Kim, Antibody-Binding Domain–Displaying HBcAg Virus-Like Particles for Antibody Immobilization, KSBB Journal 2026, 41, 87-93
Virus-like particles (VLPs) offer a versatile platform for biomedical applications owing to their uniform nanoscale architecture, structural stability, and amenability to surface engineering. In this study, we developed an antibody-binding VLP by genetically inserting the Staphylococcus aureus protein A B domain into the major immunodominant region (MIR) loop of the hepatitis B virus core antigen (HBcAg). The resulting HBcAg–B fusion protein was successfully expressed in Escherichia coli, predominantly in the soluble fraction and efficiently purified using His-tag affinity chromatography. Sucrose density gradient ultracentrifugation and transmission electron microscopy (TEM) confirmed that the HBcAg–B proteins self-assembled into well-defined VLPs with diameters of approximately 30–35 nm, comparable to native HBcAg VLPs, indicating that B domain insertion did not compromise capsid assembly or structural integrity. TEM analysis provided direct visual evidence of Fc-mediated antibody immobilization on the VLP surface, revealing multivalent antibody binding at the particle level. Surface plasmon resonance analysis further demonstrated specific and reversible antibody binding with a moderate nanomolar affinity (KD ≈ 144 nM), consistent with protein A–Fc interactions. Collectively, these results establish HBcAg–B VLPs as a structurally robust and functionally competent nanoplatform for oriented and multivalent antibody immobilization, highlighting their potential as modular carriers for therapeutic antibody delivery.
49. Ae Sol Lee and Chang Sup Kim, Harnessing cellulose-binding modules for advanced biosensing: principle, progress and perspectives, Cellulose, Accepted [IF=4.6 JCR 상위 10% 이내]
Cellulose, the most abundant natural polymer, has emerged as a sustainable platform for biosensing owing to its renewability, low cost, and biocompatibility. However, conventional probe immobilization methods on cellulose are limited by uncontrolled orientation, instability, and poor reproducibility. To overcome these barriers, cellulose-binding modules (CBMs), non-catalytic domains of carbohydrate-active enzymes, have been engineered as versatile molecular anchors. Recent studies have demonstrated that CBM fusions with enzymes, antibodies, nanobodies, nucleic acids, and reporter proteins enable oriented, high-density, and stable immobilization on cellulose scaffolds, thereby transforming cellulose from a passive carrier into an active, programmable substrate. CBM-based biosensors have been successfully applied in colorimetric, fluorescent, electrochemical, and phage-assisted platforms, achieving detection ranges from femtomolar protein biomarkers to single-colony bacterial pathogens, as well as clinically relevant monitoring of metabolites such as glucose, uric acid, and lactate. These advances underscore the potential of CBM fusion technology to improve sensitivity, reproducibility, and robustness compared to conventional cellulose-based assays. Despite these promising developments, challenges remain in clinical validation, environmental stability, and integration into portable diagnostic devices. Addressing these issues through protein engineering, nanostructured cellulose materials, and device-level innovation is expected to accelerate the translation of CBM-enabled biosensors into practical applications for healthcare, environmental monitoring, and food safety.
48. Hyo Won Kim+, Jae Hwan Shin+, Ae Sol Lee, Hyo Jeong Yang, Hwa Hui Shin, Jong Pil Park*, and Chang Sup Kim*, Electrochemical Biosensor Based on Cellulose Nanocrystals Functionalized with Peptide–Module Probes for Sensitive Detection of Influenza Virus Hemagglutinin, Talanta 2026, 308, 129855 [IF=6.7 JCR 상위 12.2%]
The high transmissibility and rapid evolution of the influenza virus have contributed to its global spread. Consequently, there is an urgent need for versatile and advanced approaches that enable rapid biosensor development for effective influenza virus detection and control. In this study, we developed a sensitive electrochemical biosensor in which a fusion of cellulose-binding modules and virus-binding peptide (CBM31-VBP1) was site-specifically immobilized on a cellulose nanocrystal (CNC)-coated electrode. The CBM31-VBP1/CNC system enhanced electrical conductivity, porosity, electroactive surface area, and active site availability for H5N1 recognition. The sensing mechanism operated via signal suppression, with the reduction in peak current driven by specific interactions between immobilized virus-binding peptides on the CNC-coated Au electrode and viral hemagglutinin (HA) proteins. The biosensor enabled sensitive detection of H5N1 hemagglutinin (HA), achieving a limit of detection (LOD) of 3.16 nM. The CBM31-guided immobilization strategy established a stable and oriented biointerface that facilitated reliable virus recognition. In addition, the biosensor demonstrated excellent storage stability, maintaining activity for 14 days at 4 °C. Moreover, low concentrations of H5N1 HA in virus transport medium were reliably detected. These findings highlight the CBM31-VBP1/CNC platform as a promising biointerface engineering strategy for sensitive and accurate detection of influenza virus, with potential to expand point-of-care diagnostic applications to diverse diseases.
Microneedle (MN) patches comprise a promising platform for transdermal delivery of
macromolecular therapeutics. However, achieving sufficient mechanical strength for skin penetration while maintaining high biocompatibility and efficient antibody loading remains a major challenge. In this study, we designed and developed a core–shell structured hydrogel MN patch composed of a silk fibroin core and a protein-based shell layer for antibody loading and potential transdermal release. The latter was constructed using a fusion protein consisting of the B and C domains of Staphylococcus aureus protein A (BC) and a tyrosine-rich mussel adhesive protein (MAP), thereby enabling antibody binding via the BC domains. By harnessing biomimetic design strategies, the BC-MAP shell facilitates antibody immobilization via specific affinity interactions, while the silk fibroin core provides substantial mechanical strength: the MN patch demonstrated a penetration force approximately 4.2 times greater than that required to pierce porcine skin. Collectively, our core-shell-structured hydrogel MN patch is a promising platform for transdermal antibody delivery.
46. Hyo Won Kim+, Ae Sol Lee+, and Chang Sup Kim*, Electrochemical biosensors based on the 3D immobilization of capture probes for influenza virus detection, RSC Adv. 2025, 15, 28565 [IF=6.1]
Influenza viruses pose a significant global health threat, particularly to vulnerable groups such as young children, the elderly, and individuals with underlying health conditions. Accurate and early detection is vital for effective disease management and the prevention of viral transmission. However, traditional diagnostic methods, including viral cultures, rapid antigen detection, and polymerase chain reaction, often face limitations associated with their sensitivity, turnaround time, cost, and/or accessibility, which hinder their effectiveness in real-world settings. Electrochemical biosensors have recently gained attention as innovative diagnostic tools because they deliver highly sensitive and specific results quickly, making them ideal for point-of-care testing. Incorporating three-dimensional (3D) structured materials can enhance biosensor performance by expanding the binding surface area for biorecognition probes and optimizing signal transduction mechanisms. This review highlights the current understanding of influenza viruses and presents the latest developments in electrochemical biosensing technologies. We emphasize the integration of materials such as metal nanoparticles, carbon-based materials, and metal–organic and covalent–organic framework-based materials that can provide 3D surfaces. These strategies enable the sensitive and selective detection of multiple influenza strains. The development of 3D probe immobilization technologies and biosensor engineering has shown promise for practical clinical implementation and large-scale diagnostic use, potentially contributing to improved influenza surveillance and public health outcomes.
45. Ae Sol Lee+, Hye Ryoung Heo+, Young Hoon Song+, Chang Sup Kim*, Jeong Hyun Seo*, and Hyung Joon Cha, Exploiting glycan arrays and biosensors to diagnose and understand diseases, Biotechnol. Adv. 2025, 83, 108635 [IF=14.1 JCR 상위 5% 이내]
Aberrant glycosylation patterns, often observed in disease states compared to those in normal states, are involved in disease-related processes such as cancer cell development and metastasis. Analyzing glycan-associated interactions could provide promising avenues for disease detection and therapeutic development. Over the past two decades, glycan arrays and biosensors have become powerful analytical tools for characterizing glycan-associated interactions, screening functional glycans, and detecting glycan-related diseases. This review aims to describe glycan source preparation and immobilization methods used to construct glycan arrays and biosensors. We summarize methods for obtaining glycans from natural sources and for chemical and enzymatic synthesis. In particular, we cover a method of immobilizing DNA, proteins, and lipids that mimic cell-surface glycoconjugates. Finally, we discuss the biomedical applications of glycan arrays and biosensors, including identification of cancer-specific glycan biomarkers, evaluation of glycan-induced antibody responses in cancer, cancer detection, analysis of glycan-binding specificities of pathogens and their toxins, pathogen detection, and drug discovery.
44. Young Hoon Song, Hye Ryoung Heo, Ae Sol Lee, Chang Sup Kim*, and Jeong Hyun Seo*, Cytotoxic effects of particulate matter on cell growth and metabolism of green fluorescent protein-expressing Escherichia coli, Korean J. Chem. Eng. 2025, 42, 2123-2129 [IF=3.2]
The toxicity of sulfate (SO4−2) and ammonium (NH4+), key components of fine dust, on living organisms was investigated using recombinant green fluorescent protein (GFP)-expressing Escherichia coli as a bioindicator. The effects of individual and mixed particulate matter (PM) compounds, including CuSO₄, (NH₄)₂SO₄, and NH₄Cl, were evaluated by measuring the optical density and GFP fluorescence intensity. Escherichia coli growth was inhibited by the individual compounds at specific thresholds, with CuSO₄ being most toxic at as low as 3.8 mM. Synergistic effects were observed with mixed compounds, markedly reducing growth and fluorescence even at lower concentrations. Notably, a mixture of the three at their sub-lethal individual concentrations completely halted bacterial growth after 2 h of incubation. CuSO₄ was a more potent inhibitor than (NH₄)₂SO₄ and NH₄Cl. These findings highlighted the importance of analyzing the individual and synergistic effects of PM components.
43. Kyeong Rok Kim†, Ae Sol Lee†, Hye Ryoung Heo, So-Young Park*, and Chang Sup Kim*, Bioinspired synthesis of virus-like particle-templated thin silica-layered nanocages with enhanced biocompatibility and cellular Uptake as drug delivery carriers, Colloids Surf. B Biointerfaces 2025, 247, 114418 [IF=5.4, 상위 10% 이내]
The bioinspired synthesis of virus-like silica nanoparticles in biomedical applications makes it possible to utilize the cellular delivery capabilities of viruses while minimizing the cytotoxicity of inorganic silica. In this study, we developed a diatom-inspired method for synthesizing silica-layered nanocages utilizing R5 peptide-functionalized virus-like particles (VLPs). R5 peptides were genetically inserted into the F-G loop of human papillomavirus 16 L1 proteins (HPV16 L1-R5). HPV16 L1-R5 was self-assembled into VLPs under an acidic pH similar to native ones and exhibited ~65% drug encapsulation efficiency. The HPV16 L1-R5 VLP@silica nanocages (SiNPs) were synthesized through diatom-inspired silicification of HPV16 L1-R5 VLPs via intermolecular interaction of the R5 peptide and polyol. HPV16L1-R5 VLP@SiNPs displayed uniform, monodisperse particles with approximately 10 nm silica layer compared to HPV16 L1-R5 VLPs. HPV16 L1-R5 VLP@SiNPs showed high biocompatibility at high concentrations, unlike commercial mesoporous SiNPs. Furthermore, the virus-like HPV16 L1-R5 VLP@SiNPs resulted in approximately 2.5-fold increased cellular uptake efficiency compared to commercial mesoporous SiNPs. These results suggest that the thin silica layer on HPV16 L1-R5 VLPs retains cellular delivery capacity while reducing cytotoxicity. Our strategy presents an innovative method for synthesizing virus-like nanoparticles in biomedical applications, enhancing cellular delivery capacity and biocompatibility.
Enzyme-linked immunosorbent assays (ELISA) have been widely used to detect disease-related antigens in clinical and research laboratories. One of the main drawbacks of ELISA is the utilization of physical adsorption for immobilizing antibodies on a surface, causing low sensitivity, reproducibility, and precision. In this study, we applied a BC-MAP linker composed of antibody-immobilizing BC domains of protein A and surface-adhesive mussel adhesive protein (MAP) to an ELISA platform to overcome these limitations. The performance of ELISA using BC-MAP linker was compared with that of untreated ELISA. BC-MAP proteins were reproducibly coated to the surface while exposing BC domains, resulting in twofold higher sensitivity and improved reproducibility of ELISA compared to the untreated ELISA utilizing physical adsorption of antibodies. Thus, the proposed method could be successfully used in ELISA platforms to diagnose and manage diseases.
Bacteriophages, also known as phages, are viruses that replicate in bacteria and archaea. Phages were initially discovered as antimicrobial agents, and they have been used as therapeutic agents for bacterial infection in a process known as “phage therapy.” Recently, phages have been investigated as functional nanomaterials in a variety of areas, as they can function not only as therapeutic agents but also as biosensors and tissue regenerative materials. Phages are nontoxic to humans, and they possess self-assembled nanostructures and functional properties. Additionally, phages can be easily genetically modified to display specific peptides or to screen for functional peptides via phage display. Here, we demonstrated the application of phage nanomaterials in the context of tissue engineering, sensing, and probing.
Bioinspired enzyme encapsulation technologies have received increasing attention in sustainable development owing to the enzyme protection from external stressors while mimicking the cellular environment and structure. In this study, we developed a diatom-inspired synthesis of ultrathin silica-layered nanoparticles directed by silica-forming R5 peptide- and carbonic anhydrase (CA)-functionalized micelles. Each CA and R5 peptide was covalently conjugated with N-hydroxysuccinimide (NHS)-ester-modified hydrophilic ends of the triblock copolymer F127 (F127–CA and F127–R5). F127–CA/R5 micelles were prepared by controlling the molar ratio of F127–CA and F127–R5. F127–CA/R5 micelle@silica nanoparticles (SiNPs) were synthesized through R5 peptide-catalyzed silicification of the F127–CA/R5 micelle in a two-phase system. F127–CA/R5 micelle@SiNPs exhibited uniform and monodisperse particles with a size of 17 nm, indicating the formation of a ~1.5-nm ultrathin and mesoporous silica layer compared with F127–CA/R5 micelle. F127–CA/R5 micelle@SiNPs exhibited almost identical KM and kcat values in CO2 hydration activity compared with the free enzyme. In addition, F127–CA/R5 micelle@SiNPs also showed enhanced stability compared to free ngCA under the operation condition of CO2 capture and sequestration with good storage stability. These results indicated that an ultrathin and mesoporous silica layer on the micelle could protect enzymes from external environments while minimizing limited mass transfer. Thus, our strategy could offer a new direction for enzyme-based green chemistry in practical applications, providing enhanced mass transfer and stability.
Virus-like nanoparticles (VLPs) are natural polymer-based nanomaterials that mimic viral structures through the hierarchical assembly of viral coat proteins, while lacking viral genomes. VLPs have received enormous attention in a wide range of nanotechnology-based medical diagnostics and therapies, including cancer therapy, imaging, and theranostics. VLPs are biocompatible and biodegradable and have a uniform structure and controllable assembly. They can encapsulate a wide range of therapeutic and diagnostic agents, and can be genetically or chemically modified. These properties have led to sophisticated multifunctional theranostic platforms. This article reviews the current progress in developing and applying engineered VLPs for molecular imaging, drug delivery, and multifunctional theranostics in cancer research.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). The high human-to-human transmission and rapid evolution of SARS-CoV-2 have resulted in a worldwide pandemic. To contain SARS-CoV-2, it is essential to efficiently control the transmission of the virus through the early diagnosis of infected individuals, including asymptomatic people. Therefore, a rapid and accurate assay is vital for the early diagnosis of SARS-CoV-2 in suspected individuals. In this study, we developed a colorimetric lateral flow immunoassay (LFIA) in which a CBP31-BC linker was used to immobilize antibodies on a cellulose membrane in an oriented manner. The developed LFIA enabled sensitive detection of cultured SARS-CoV-2 in 15 min with a detection limit of 5 × 104 copies/mL. The clinical performance of the LFIA for detecting SARS-CoV-2 was evaluated using 19 clinical samples validated by reverse transcription-polymerase chain reaction (RT–PCR). The LFIA detected all the positive and negative samples accurately, corresponding to 100% accuracy. Importantly, patient samples with low viral loads were accurately identified. Thus, the proposed method can provide a useful platform for rapid and accurate point-of-care testing of SARS-CoV-2 in infected individuals to efficiently control the COVID-19 pandemic.
Bone graft materials have been mainly developed based on inorganic materials, including calcium phosphate. However, these graft materials usually act as osteoconductive rather than osteoinductive scaffolds. To improve bone reconstruction, the combination of several materials have been proposed. However, there are still no alternatives that can completely replace the existing animal-derived bone graft materials. In this work, a marine-inspired biomineral complex was suggested as a potential bone graft material. The proposed biosilicified coccolithophore-derived coccoliths using bioengineered mussel adhesive protein show osteopromotive ability through the synergistic effects of osteoconductivity from calcium carbonate and osteoinductivity from silica. Its possibility of use as a bone substitute was determined by evaluating the in vitro osteogenic behaviors of multipotent mesenchymal stem cells and in vivo bone regeneration in rat calvarial defect model. Therefore, the marine-inspired biomineral complex developed in this study could be successfully used for bone tissue engineering.
36. Hye Ryoung Heo, Kye Il Joo, Jeong Hyun Seo, Chang Sup Kim*, and Hyung Joon Cha*, Glycan chip based on structure-switchable DNA linker for on-chip biosynthesis of cancer-associated complex glycans, Nat. Commun. 2021, 12, 1395 [IF=16.6, JCR 상위 5% 이내]
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 structureswitchable DNA linker-based glycan chip platform for on-chip complex glycan biosynthesis and glycan-involved applications.
An advanced paper-based LFIA using engineered cellulose-binding protein linker fused with antibody-binding domains was developed to resolve problems associated with conventional LFIA strip, including the limitation of quantitative analysis and low sensitivity. CBP31-BC linker was prepared by genetically fusing the cellulosebinding modules of family 3 (CBM3) and family 1 (CBM1) with antibody-binding B and C domains of Protein A. Cellulose-binding analysis showed that the addition of two CBMs led to high cellulose-binding capacity in CBP31-BC. Transmission electron microscopy and quartz crystal microbalance analyses demonstrated that the cellulose-binding ability of CBP31-BC enabled the BC domain to be efficiently exposed, allowing about 6-fold higher antibody-binding efficiency (~32.5 %) than that (~5.5 %) of sole BC. To evaluate the feasibility of the CBP31-BC linker-employed LFIA platform, an immunoassay of prostate-specific antigen (PSA) was performed as a model reaction. The CBP31-BC-employed paper-based LFIA detected PSA at levels as low as 0.25 ng/mL in 20 min, which is about 10-fold more sensitive to conventional LFIAs that use simple physical adsorption. CBP31- BC-employed LFIA also exhibited a lower detection range of 0.25–2.5 ng/mL when compared to conventional LFIAs. These results might be due to efficient oriented immobilization of the capture antibody by CBP31-BC linker on cellulose membrane. Thus, our developed CBP31-BC linker-employed paper-based LFIA platform holds great promise for the detection of target markers due to significantly enhanced sensitivity.
34. Jong Min Yang+, Gi Eob Kim+, Kyeong Rok Kim, and Chang Sup Kim*, Expression and purification of the full-length N-acetylgalactosaminyltransferase and galactosyltransferase from Campylobacter jejuni in Escherichia coli, Enzyme Microb. Technolol. 2020, 135, 109489 [IF=3.4]
The successful enzymatic synthesis of various ganglioside-related oligosaccharides requires many available glycan-processing enzymes. However, the number of available glycan-processing enzymes remains limited. In this study, the full-length CgtA43456 (β-(1→4)-N-acetylgalactosaminyltransferase) and CgtB11168 (β-(1→3)- galactosyltransferase) were successfully produced from Escherichia coli through the optimization of E. coli–preferable codon usage, selection of E. coli strain, and use of the molecular chaperone GroEL-GroES (GroEL/ES). The CgtA43456 enzyme was produced as a soluble form in E. coli C41(DE3) co-expressed with codon-optimized CgtA43456 and GroEL/ES. However, soluble CgtB11168 was well expressed in E. coli C41(DE3) with only the codon-optimized CgtB11168. Rather, when co-expressed with GroEL/ES, total production of CgtB11168 was reduced. Using immobilized-metal affinity chromatography, the CgtA43456 and CgtB11168 proteins were obtained with approximately 75–78 % purity. The purified CgtA43456 showed a specific activity of 21 mU/mg using UDP-N-acetylgalactosamine and GM3 trisaccharide as donor and acceptor, respectively. The purified CgtB11168 catalyzed the transfer of galactose from UDP-Gal to GM2 tetrasaccharide with a specific activity of 16 mU/mg. We propose that they could be used as catalysts for enzymatic synthesis of GM1 ganglioside–related oligosaccharides.
33. Dong Gyun Kang+, Chang Sup Kim+, and Hyung Joon Cha*, Co-expression of CMP-sialic acid transporter reduces N-glycolylneuraminic acid levels of recombinant glycoproteins in Chinese hamster ovary cells, Biotechnol. Bioeng. 2019, 116, 2815-2822 [IF=3.8]
Recombinant glycoproteins expressed in Chinese hamster ovary (CHO) cells contain two forms of sialic acids; N-acetylneuraminic acid (Neu5Ac) as a major type and N-glycolylneuraminic acid (Neu5Gc) as a minor type. The Neu5Gc glycan moieties in therapeutic glycoproteins can elicit immune responses because they do not exist in human. In the present work, to reduce Neu5Gc levels of recombinant glycoproteins from CHO cell cultures, we coexpressed cytidine-5'-monophosphate-sialic acid transporter (CMP-SAT) that is an antiporter and transports cytosolic CMP-sialic acids (both forms) into Golgi lumen. When human erythropoietin was used as a target human glycoprotein, coexpression of CMP-SAT resulted in a significant decrease of Neu5Gc level by 41.4% and a notable increase of Neu5Ac level by 21.2%. This result could be reasonably explained by our hypothesis that the turnover rate of Neu5Ac to Neu5Gc catalyzed by CMP-Neu5Ac hydroxylase would be reduced through facilitated transportation of Neu5Ac into Golgi apparatus by coexpression of CMP-SAT. We confirmed the effects of CMP-SAT coexpression on the decrease of Neu5Gc level and the increase of Neu5Ac level using another glycoprotein human DNase I. Therefore, CMP-SAT coexpression might be an effective strategy to reduce the levels of undesired Neu5Gc in recombinant therapeutic glycoproteins from CHO cell cultures.
32. Chang Sup Kim+, Hye Ryoung Heo+, Jeong Hyun Seo, and Hyung Joon Cha*, On-chip biosynthesis of GM1 pentasaccharide-related complex glycans, Chem. Comm. 2019, 55, 71-74 [IF=4.9]
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.
31. Jong Min Yang+, Kyeong Rok Kim+, and Chang Sup Kim*, Biosensor for rapid and sensitive detection of influenza virus, Biotechnol. Bioproc. E. 2018, 23, 371-382 [IF=3.2]
Influenza viruses continue to threaten human life, causing considerable damage socially and economically. To reduce influenza-related morbidity and mortality, there is an immediate requirement to develop efficient and effective tools to detect the virus. Several methods are currently employed for diagnosing influenza infections in humans, including viral culture, polymerase chain reaction (PCR), and immunoassay. In addition, biosensors are being developed to improve the limitations of the conventional methods. In this article, we review the current progress in investigative techniques, including the development of biosensors having high sensitivity and selectivity and shorter detection time.
30. Young Hoon Song, Jeong Chan Park, Chang Sup Kim, Dong Soo Hwang, Hyung Joon Cha, and Jeong Hyun Seo*, Sucrose-calcium complexation for the durable biomass pellet, Biotechnol. Bioproc. E. 2018, 23, 341-348 [IF=3.2]
Due to their environmental friendliness, woodbased biomass pellets are widely used as conventional fuel sources in daily life and in various industries. Durability and proper mechanical strength are important quality factors for practical applications of biomass pellets as they enable their easy handling, transportation, and storage. In the present study, to increase mechanical strength of sawdust biomass pellet fuels, sucrose was employed as a main binder material. In addition, calcium ions were included as cross-linkers, which improved capability of modified binders. Even though sucrose alone enabled production of pellets with comparatively high compressive strength, addition of several calcium-containing substances further improved mechanical properties of sawdust pellets. Interestingly, we found that a combination of sucrose with CaCl2 (acidic blended solution) decreased, whereas addition of CaO or Ca(OH)2 (basic blended solution) considerably enhanced pellet strength. Thus, we concluded that calcium ions are able to form stable complexes with sucrose at basic pH levels ( >10). Therefore, materials incorporating sucrose-calcium complexes can be successfully used as eco-friendly novel binders for the construction of durable biomass pellet fuels. Furthermore, their applications can be extended to formulations of nutritional or pharmaceutical substances.
29. Jeong Chan Park, Do Hyeon Kim, Chang Sup Kim, and Jeong Hyun Seo*, R5 peptide-based silicification using methyltrimethoxysilane, Biotechnol. Bioproc. E. 2018, 23, 11-15 [IF=3.2]
We examined the performance of methyltrimethoxysilane (MTMS), a precursor of silicic acid, in the process of biosilicification induced by the R5 peptide from Cylindrotheca fusiformis. Recombinant GFP-R5 fusion protein was produced by Escherichia coli cultured at 25°C as a soluble and functional formation, but not at 37°C. MTMS-based biosilica deposits had a larger average diameter compared to tetraethyl orthosilicate (TEOS)-based deposits. Reducing phosphate concentration in the buffer system led to a decrease in the size of MTMS-based biosilica. These results provide insight into the surface modification of biosilica, and control of biosilica particle size, when using hydrophobic precursors such as MTMS.
28. Yun Kee Jo, Bong‐Hyuk Choi, Chang Sup Kim*, and Hyung Joon Cha*, Diatom-inspired silica nanostructure coatings with controllable microroughness using an engineered mussel protein glue to accelerate bone growth on titanium-based implants, Adv. Mater. 2017, 29(46), 1704906-1704914 [IF=29.4, JCR 상위 5% 이내]
Silica nanoparticles (SiNPs) have been utilized to construct bioactive nanostructures comprising surface topographic features and bioactivity that enhances the activity of bone cells onto titanium-based implants. However, there have been no previous attempts to create microrough surfaces based on SiNP nanostructures even though microroughness is established as a characteristic that provides beneficial effects in improving the biomechanical interlocking of titanium implants. Herein, a protein-based SiNP coating is proposed as an osteopromotive surface functionalization approach to create microroughness on titanium implant surfaces. A bioengineered recombinant mussel adhesive protein fused with a silica-precipitating R5 peptide (R5- MAP) enables direct control of the microroughness of the surface through the multilayer assembly of SiNP nanostructures under mild conditions. The assembled SiNP nanostructure significantly enhances the in vitro osteogenic cellular behaviors of preosteoblasts in a roughness-dependent manner and promotes the in vivo bone tissue formation on a titanium implant within a calvarial defect site. Thus, the R5-MAP-based SiNP nanostructure assembly could be practically applied to accelerate bone-tissue growth to improve the stability and prolong the lifetime of medical implantable devices.
27. Chang Sup Kim+, Yun Jung Yang+, So Yeong Bahn, and Hyung Joon Cha*, A bioinspired dual-crosslinked tough silk protein hydrogel as a protective biocatalytic matrix for carbon sequestration, NPG Asia Mater. 2017, 9, e391 [IF=9.7]
The development of carbonic anhydrase (CA)-based materials for the environment-friendly sequestration of carbon dioxide (CO2) under mild conditions would be highly valuable for controlling emissions to the environment and for producing value-added chemicals. Here, a highly tough and stable CA-encapsulating silk protein hydrogel was developed as a robust biocatalyst for CO2 sequestration through a bioinspired dual-crosslinking strategy that employed photoinduced dityrosine chemical crosslinking followed by dehydration-mediated physical crosslinking. The target enzyme was efficiently encapsulated in the silk hydrogel with ~ 60% retention of the activity of free CA, and the encapsulated CA exhibited excellent overall multi-use, storage and thermal stabilities. The dual-crosslinked CA-encapsulating silk hydrogel exhibited a significant compressive modulus, which surpassed the moduli of most traditional and double-network hydrogels as well as those of enzyme-encapsulated hydrogels. This hydrogel also showed high resiliency and elasticity and outstanding structural stability. Importantly, the dual-crosslinked CA-encapsulating silk hydrogel facilitated the sequestration of CO2 into calcium carbonate with high CO2 hydration activity. Thus, the unique combination of bioinspired dual-crosslinking with silk fibroin protein and CA enzyme demonstrates the successful application of this protein hydrogel as a promising biocatalyst for CO2 sequestration by showing high activity, strong mechanical properties and outstanding structural stability.
26. Do Hyeon Kim, Jeong Chan Park, Go Eun Jeon, Chang Sup Kim, and Jeong Hyun Seo*, Effect of the size and shape of silver nanoparticles on bacterial growth and metabolism by monitoring optical density and fluorescence intensity, Biotechnol. Bioproc. E. 2017, 22, 210-217 [IF=3.2]
In this study, we demonstrate the antibacterial activity of silver nanoparticles (AgNPs), depending on their size and shape, on green fluorescent protein (GFP)- expressing E. coli, which provides a facile, rapid, and noninvasive monitoring system. By measuring optical density and fluorescence intensity in the recombinant E. coli, we found that smaller sized plate-shaped AgNPs presented higher antibacterial activity than larger sized, cubic and spherical AgNPs. In the case of 10 nm spherical AgNPs, the optical density was detectable at 15 ng/mL after 12 h incubation, but the fluorescence intensity was not. On the other hand, smaller-sized AgNPs showed higher toxicity than plate-shaped AgNPs based on the measurement of the optical density and fluorescence intensity. The combined analysis of optical density and fluorescence intensity may be helpful for understanding the effect of various materials, including nano- and organic materials, on recombinant bacteria.
25. Jeong Chan Park, Gyeong Tae Lee, Chang Sup Kim, and Jeong Hyun Seo*, Effect of surface charge and ligand type of Au nanoparticles on green fluorescence protein-expressing Escherichia coli, Biotechnol. Bioproc. E. 2017, 22, 83-88 [IF=3.2]
The effects of the surface charge and ligand type of three types of Au nanoparticles (NPs), namely anionic polyethylene glycol (PEG)-Au NPs, anionic citrate (Cit)-Au NPs, and cationic branched polyethylenimine (bPEI)-Au NPs, on green fluorescent protein (GFP)- expressing Escherichia coli were evaluated through the combined analysis of optical density (OD) and fluorescence intensity (FI). OD and FI can provide information about cell growth and metabolism of bacteria, respectively. The results demonstrated that PEG- and Cit-Au NPs had no major effects on the OD and FI of GFP-expressing bacteria. However, it was found that Cit-Au NPs may slightly influence cell metabolism at higher concentrations, although it is necessary to perform further in-depth study to clarify this issue. Cationic bPEI-Au NPs showed significant effects on cell density and metabolism of E. coli, with an especially strong effect on metabolism. The combined analysis of OD and FI may be useful for monitoring the effects of a wide range of nanomaterials on microorganisms.
24. Kyeoung Rok Kim, Young Hoon Song, Jeong Hyun Seo, Dong Gyun Kang, and Chang Sup Kim*, Effect of culture conditions on the whole cell activity of recombinant Escherichia coli expressing periplasmic organophosphorus hydrolase and cytosolic GroEL/ES chaperone, Biotechnol. Bioproc. E. 2016, 21, 502-507 [IF=3.2]
Specific whole cell activity strongly affects sensitivity and detection limit of whole cell-based biosensors. Previously, we developed recombinant Escherichia coli coexpressing periplasmic organophosphorus hydrolase (OPH) and cytosolic chaperone GroEL-GroES (GroEL/ES). In present work, we investigated the effect of culture conditions on whole cell OPH activity. Especially, the whole cell OPH activity was significantly affected by the concentration of tetracycline that is an inducer for chaperone GroEL/ES. When cultured at 20°C for 31 h in M9 medium containing 1 mM IPTG, 50 ng/mL tetracycline, and 500 µM CoCl2, the recombinant E. coli exhibited a specific whole cell OPH activity (U/OD600) of ~0.55, which is 2.6-fold higher than that of recombinant E. coli cultured as previously described conditions. In addition, recombinant cells showed adequate storage stability for 1 week with 100% of original response. Finally, the improved activity and adequate stability in the whole cell biocatalyst will contribute to sensitivity, detection time, and stability of a whole cell-based biosensor for the detection of toxic organophosphates.
23. Byung Hoon Jo+, Chang Sup Kim+, Yun Kee Jo+, Hogyun Cheong+, and Hyung Joon Cha*, Recent developments and applications of bioinspired silicification, Korean J. Chem. Eng. 2016, 33, 1125-1133 [IF=2.7]
Bioinspired synthesis of silica has attracted attention from a wide range of researchers as novel route for fabrication of various nanomaterials. Proteins including silaffins and silicateins as well as polyamines from marine diatoms and sponges are key biomolecules in these biomimetic silicification processes. These methods allow silica mineralization from various silica precursors under mild, biologically compatible conditions in an unprecedentedly fast and facile manner. Notably, the silica polycondensation entails the concomitant encapsulation of other molecules in the reaction solutions. Due to the efficient encapsulation and synergetic effects brought by the encapsulated molecules and the characteristics of biomimetic silica synthesis as well as the mechanical and chemical properties of silica itself, the silica-biomolecule nanocomposites have broad applications in biocatalysis, biosensor, and biomedical areas. Introduction and combination of novel template, precursors, inorganics, or enzymes with the previously used strategies will allow construction of more efficient, purpose-optimized silica nanomaterials with controlled size, composition, and morphology.
22. Dong Gyun Kang, Jeong Hyun Seo, Byung Hoon Jo, Chang Sup Kim, Suk Soon Choi, and Hyung Joon Cha*, Versatile signal peptide of Flavobacterium-originated organophosphorus hydrolase for efficient periplasmic translocation of heterologous proteins in Escherichia coli, Biotechnol. Progr. 2016, 32, 848-854 [IF=2.9]
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.
21. Hwa Hui Shin, Jeong Hyun Seo, Chang Sup Kim, Byeong Hee Hwang, and Hyung Joon Cha*, 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, 79, 398-405 [IF=12.6, JCR 상위 5% 이내]
Life-threatening diarrheal cholera is usually caused by water or food contaminated with cholera toxinproducing 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.
20. Young Hoon Song, Chang Sup Kim, and Jeong Hyun Seo*, Noninvasive monitoring of environmental toxicity through green fluorescent protein expressing Escherichia coli, Korean J. Chem. Eng. 2016, 33, 1331-1336 [IF=2.7]
The facile and fast monitoring of cellular response against the environmental stresses is crucial for understanding the effect of environmental toxicity in living organisms. It would be better to look at the overall cell growth and find the easy monitoring system. Green fluorescent protein (GFP) has advantageous when used as a reporter protein of cellular stress responses in Escherichia coli since it makes possible for non-invasive monitoring of GFP in vivo without affecting cell metabolism. Here, we compared the environmental toxicities of chemical pollutants such as ethanol, phenol, para-formaldehyde, paraben, and triclosan using GFP expressing E. coli for easy monitoring. For this, cell density and fluorescence intensity were analyzed from cells incubated with diverse concentrations of harmful chemicals, and we were able to look at the level of toxicity and to characterize critical concentration on the cells by simply measuring GFP fluorescence intensity. From the analysis of cellular stress response, we confirmed that all of chemical pollutants act on cell growth and cell metabolism as measuring cell density and fluorescent intensity of GFP. Also, we found that some chemical pollutants (ex. paraformaldehyde and phenol) could effect on bacterial cells differently depending on the concentration. In addition, from comparative analysis for quantification, we were able to deduce the concentration for unknown ethanol toxicity, which was not conducted at the concentration. In conclusion, the degree of toxicity for each chemical pollutant could be estimated or evaluated and it should be noted that this system would be useful for monitoring the toxicity of chemical pollutants as a non-invasive monitoring system.
19. Chang Sup Kim, Jeong Hyun Seo, and Hyung Joon Cha*, Functional Characterization of Vibrio cholera O1 WbeW enzyme responsible for initial reaction in O antigen biosynthesis, Biotechnol. Bioproc. E. 2015, 20, 980-987 [IF=3.2]
Vibrio cholerae O1 employs the ATP-binding cassette (ABC) transporter-dependent pathway for O antigen biosynthesis. Different from highly studied Klebsiella pneumoniae and Escherichia coli, it was reported that initial reaction of O antigen biosynthesis in V. cholerae O1 may be involved in WbeW protein, which is predicted to be a galactosyltransferase. In this work, we report expression and characterization of WbeW enzyme. WbeW was expressed as membrane-associated form in E. coli and it was obtained with high purity. The enzyme had a function of transferring Gal-1-P from UDP-Gal to Und-P, implying that initial glycan of O antigen in V. cholerae O1 can be composed of a Gal residue.
18. Intae Kim, Geon Hwee Kim, Chang Sup Kim, Hyung Joon Cha, and Geunbae Lim*, Optical detection of paraoxon using single-walled carbon nanotube films with attached organophosphorus hydrolase-expressed Escherichia coli, Sensors 2015, 15, 12513-12525 [IF=3.9]
In whole-cell based biosensors, spectrophotometry is one of the most commonly used methods for detecting organophosphates due to its simplicity and reliability. The sensor performance is directly affected by the cell immobilization method because it determines the amount of cells, the mass transfer rate, and the stability. In this study, we demonstrated that our previously-reported microbe immobilization method, a microbe-attached single-walled carbon nanotube film, can be applied to whole-cell-based organophosphate sensors. This method has many advantages over other whole-cell organophosphate sensors, including high specific activity, quick cell immobilization, and excellent stability. A device with circular electrodes was fabricated for an enlarged cell-immobilization area. Escherichia coli expressing organophosphorus hydrolase in the periplasmic space and single-walled carbon nanotubes were attached to the device by our method. Paraoxon was hydrolyzed using this device, and detected by measuring the concentration of the enzymatic reaction product, p-nitrophenol. The specific activity of our device was calculated, and was shown to be over 2.5 times that reported previously for other whole-cell organophosphate sensors. Thus, this method for generation of whole-cell-based OP biosensors might be optimal, as it overcomes many of the caveats that prevent the widespread use of other such devices.
17. Yun Jung Yang, Chang Sup Kim, Bong-Hyuk Choi, and Hyung Joon Cha*, Mechanical durable and biologically favorable protein hydrogel based on elastic silklike protein derived from sea anemone, Biomacromolecules 2015, 16, 3819-3826 [IF=6.2]
As biodegradable scaffolds, protein hydrogels have considerable potential, particularly for bioartificial organs and threedimensional space-filling materials. However, their low strength and stiffness have been considered to be limitations for enduring physiological stimuli. Therefore, protein hydrogels have been commonly utilized as delivery vehicles rather than as supporting materials. In this work, sea anemone tentacle-derived recombinant silk-like protein (aneroin) was evaluated as a potential material for a mechanically durable protein hydrogel. Inspired by the natural hardening mechanism, photoinitiated dityrosine cross-linking was employed to fabricate an aneroin hydrogel. It was determined that the fabricated aneroin hydrogel was approximately 10-fold stiffer than mammalian cardiac or skeletal muscle. The aneroin hydrogel provided not only structural support but also an adequate environment for cells. It exhibited an adequate swelling ability and microstructure, which are beneficial for facilitating mass transport and cell proliferation. Based on its mechanical and biological properties, this aneroin hydrogel could be used in various biomedical applications, such as cell-containing patches, biomolecule carriers, and artificial extracellular matrices.
16. Chang Sup Kim+, Jeong Hyun Seo+, Dong Gyun Kang, and Hyung Joon Cha*, Engineered whole-cell biocatalyst-based detoxification and detection of neurotoxic organophosphate compounds, Biotechnol. Adv. 2014, 32, 652-662 [IF=16.0, JCR 상위 5% 이내]
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.
15. Intae Kim, Taechang An, WooSeok Choi, Chang Sup Kim, Hyung Joon Cha, and Geunbae Lim*, Site-specific immobilization of microbes using carbon nanotubes and dieletrophoretic force for microfluidic applications, RSC Adv. 2014, 4, 1347-1351 [IF=3.9]
We developed a microbial immobilization method for successful applications in microfluidic devices. Single-walled nanotubes and Escherichia coli were aligned between two cantilever electrodes by a positive dielectrophoretic force resulting in a film of single-walled nanotubes with attached Escherichia coli. Because this film has a suspended and porous structure, it has a larger reaction area and higher reactant transfer efficiency than film attached to the substrate surface. The cell density of film was easily controlled by varying the cell concentration of the suspension and varying the electric field. The film showed excellent stability of enzyme activity, as demonstrated by measuring continuous reaction and long-term storage times using recombinant Escherichia coli that expressed organophosphorus hydrolase.
14. Jeong Hyun Seo+, Chang Sup Kim+, and Hyung Joon Cha*, Structural evaluation of GM1-related carbohydrate-cholera toxin interactions through surface plasmon resonance kinetic analysis, Analyst 2013, 138, 6924-6929 [IF=4.2]
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 Nacetylgalactosamine 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.
13. Chang Sup Kim+, Bong-Hyuk Choi+, Jeong Hyun Seo, Geunbae Lim, and Hyung Joon Cha*, Mussel adhesive protein-based whole cell array biosensor for detection of organophosphorus compounds, Biosens. Bioelectron. 2013, 41, 199-204 [IF=12.6, JCR 상위 5% 이내]
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 paraoxonhydrolyzing 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 mg/cm2 ) and cell loading (4 OD600), detected paraoxon levels as low as 5 mM with high reproducibility, and its quantitative detection range was 5–320 mM. 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.
12. Ji Eun Lee, Jeong Hyun Seo, Chang Sup Kim, Yunkyeoung Kwon, Jeong Hyub Ha, Suk Soon Choi, and Hyung Joon Cha*, A comparative study on antibody immobilization strategies onto solid surface, Korean J. Chem. Eng. 2013, 30, 1934-1938 [IF=2.7]
Antibody immobilization onto solid surface has been studied extensively for a number of applications including immunoassays, biosensors, and affinity chromatography. For most applications, a critical consideration regarding immobilization of antibody is orientation of its antigen-binding site with respect to the surface. We compared two oriented antibody immobilization strategies which utilize thiolated-protein A/G and thiolated-secondary antibody as linker molecules with the case of direct surface immobilization of thiol-conjugated target antibody. Antibody immobilization degree and surface topography were evaluated by surface plasmon resonance and atomic force microscope, respectively. Protein A/G-mediated immobilization strategy showed the best result and secondary antibody-mediated immobilization was the worst for the total immobilization levels of target antibodies. However, when considering realto-ideal ratio for antigen binding, total target antigen binding levels (oriented target antibody immobilization levels) had the following order: secondary antibody-mediated immobilization >protein A/G-mediated immobilization >direct thiol-conjugated immobilization. Thus, we confirmed that protein A/G- and secondary antibody-mediated strategies, which consider orientation of target antibody immobilization, showed significantly high antigen binding efficiencies compared to direct random immobilization method. Collectively, the oriented antibody immobilization methods using linker materials could be useful in diverse antibody-antigen interaction-involved application fields.
11. Suk Soon Choi, Hyun Min Lee, Jeong Hyub Ha, Dong Gyun Kang, Chang Sup Kim, Jeong Hyun Seo, and Hyung Joon Cha*, Biological removal of phosphate at low concentrations using recombinant Escherichia coli expressing phosphate-binding protein in periplasmic space, Appl. Biochem. Biotech. 2013, 171, 1170-1177 [IF=3.0]
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.
10. Chang Sup Kim+, Jeong Hyun Seo+, and Hyung Joon Cha*, Functional interaction analysis of GM1-related carbohydrates and Vibrio cholerae toxins using carbohydrate microarray, Anal. Chem. 2012, 84, 6884-6890 [IF=7.4]
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 (ctxAB5) 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 ctxAB5-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.
9. Im Gyu Kim, Byung Hoon Jo, Dong Gyun Kang, Chang Sup Kim, Yoo Seong Choi, and Hyung Joon Cha*, Biomineralization-based conversion of carbon dioxide to calcium carbonate using recombinant carbonic anhydrase, Chemosphere 2012, 87, 1091-1096 [IF=8.8]
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 CO2 to valuable chemicals. While purified bovine CA (BCA) has been used in previous studies, its practical utilization in CO2 conversion has been limited due to the expense of BCA preparation. In the present work, we investigated conversion of CO2 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 CO2 conversion. Recombinant a-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 CO2 hydration activity to commercial BCA significantly promoted formation of solid CaCO3 through the acceleration of CO2 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 CO2 conversion system.
8. Dong Gyun Kang, Chang Sup Kim, Jeong Hyun Seo, Im Gyu Kim, Suk Soon Choi, Jeong Hyub Ha, Soo Wan Nam, Geunbae Lim, and Hyung Joon Cha*, Coexpression of molecular chaperon enhances activity and export of organophosphorus hydrolase in Escherichia coli, Biotechnol. Progr. 2012, 28, 925-930 [IF=2.9]
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.
7. Chang Sup Kim, Yoo Seong Choi, Wooree Ko, Jeong Hyun Seo, Jieun Lee, and Hyung Joon Cha*, A mussel adhesive protein fused with the BC domain of protein A is a functional linker material that efficiently immobilizes antibodies onto diverse surfaces, Adv. Funct. Mater. 2011, 21, 4101-4108 [IF=19.0, JCR 상위 5% 이내]
The efficient immobilization of antibodies onto solid surfaces is vital for the sensitivity and specifi city 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 effi cient 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 effi ciently immobilized on BC-MAP-coated surfaces, and the immobilized antibodies interact selectively with their corresponding antigen. Quartzcrystal-microbalance analyses show that BC-MAP has excellent antibodybinding 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 effi cient 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 effi cient immobilization of antibodies onto diverse solid supports.
6. Yoo Seong Choi, Dong Gyun Kang, Seonghye Lim, Yun Jung Yang, Chang Sup Kim, and Hyung Joon Cha*, Recombinant mussel adhesive protein fp-5 (MAP fp-5) as a bulk bioadhesive and surface coating material, Biofouling 2011, 27, 729-737 [IF=2.7]
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.
5. Md. Mokarrom Hossain, Chang Sup Kim, Hyung Joon Cha, Hye Jin Lee, Amperometric detection of parathion and methyl parathion with a microhole-ITIES, Electroanal. 2011, 23, 2049-2056 [IF=3.0]
An amperometric sensor featuring a microhole-liquid/gel interface for the detection of both parathion and methyl parathion is developed on the basis of their different kinetics behavior when interacting with the enzyme organophosphorus hydrolase (OPH). OPH hydrolyzes parathion and methyl parathion producing a common product of para-nitrophenol and either diethylthio- or dimethylthio- phosphoric acid, respectively, of which all can release protons depending upon their pKa values. The detection method for both organophosphate (OP) compounds is designed to measure the current associated with the transfer of protons released from the products of OPH hydrolysis across a polarized microhole-water/polyvinylchloride-nitrophenyloctylether (PVC-NOPE) gel interface. The selective transfer of protons across the interface is tailored by the use of a proton selective ligand, ETH 1778, in the gel layer. A disposable proton selective sensor that can quantitatively analyze the OP compounds is also fabricated using simple polydimethylsiloxane microfabrication. Cyclic voltammetry and differential pulse stripping voltammetry are first utilized to characterize the transfer of protons across the microhole-water/PVC-NPOE gel interface initiated by the OPH reaction with parathion and methyl parathion and to establish a detection limit for each OP compound. In order to sequentially detect parathion and methyl parathion using a single proton selective stripsensor, a novel time-resolved detection methodology is developed based on the different catalytic kinetics of OPH with each OP analyte; the maximum peak current for the preconcentrated protons transferring back from the organic to water phase assisted by ETH 1778 increases proportionally to the concentration of each OP agent. Since the maximum peak currents for both OP analytes are observed at different reaction times it was possible to demonstrate the multiplexed analysis of both parathion and methyl parathion down to 0.5 mM using a single sensor.
4. Changho Chun, Jinmyoung Joo, Donghoon Kwon, Chang Sup Kim, Hyung Joon Cha, Myung-Sub Chung, and Sangmin Jeon*, A facile and sensitive immunoassay for the detection of alpha-fetoprotein using gold-coated magnetic nanoparticle clusters and dynamic light scattering, Chem. Commun. 2011, 47, 11047-110495 [IF=4.9]
A facile and sensitive immunoassay protocol for the detection of alpha-fetoprotein (AFP) was developed using gold-coated iron oxide magnetic nanoclusters and dynamic light scattering (DLS) methods. The increase in the average particle size due to AFP-mediated aggregation was measured using DLS, and the detection limit was better than 0.01 ng mL-1.
3. Jeong Hyun Seo, Chang Sup Kim, Hea Yeon Lee, Tomoji Kawai, and Hyung Joon Cha*, Interactive configuration through force analysis of GM1 pentasaccharide-Vibrio cholera toxin interaction, Anal. Chem. 2011, 83, 6011-6017 [IF=7.4]
Understanding of the molecular relationships in carbohydrateprotein interactions provides useful information on biological processes in living organisms and is also helpful for development of potent biomedical agents. Herein, the interaction unbinding force between GM1 pentasaccharide and Vibrio cholera toxin (ctx) proteins was measured using atomic force microscopy (AFM), which enabled us to determine the interaction of ctx holotoxin (ctxAB) with GM1 and the interactive formation. First, the interaction force measured between A and B subunits (ctxActxB) was 184.2 ( 4.5 pN, and the unbinding forces were evaluated to confirm the role of ctxA in ctxAB complex formation and were determined to be 443.7 ( 7.5 and 535.7 ( 25.9 pN for GM1ctxB and GM1ctxAB complexes, respectively. The force difference of ∼90 pN between GM1ctxB and GM1ctxAB might be due to the formation of the cholera toxin complex. Importantly, from the analogue analyses, we understand how structural and binding positional differences in complex carbohydrates affect the interaction with protein and surmise that the GM1ctxAB complex makes a “two-finger grip” formation through the conformational change of a flexible carbohydrate. In conclusion, using AFM force analysis, we successfully quantified and characterized the interactive configuration of carbohydrateprotein molecules.
2. Md. Mokarrom Hossain, Shaikh Nayeem Faisal, Chang Sup Kim, Hyung Joon Cha, Sang Cheol Nam, Hye Jin Lee*, Amperometric proton selective strip-sensors with a microelliptic liquid/gel interface for organophosphate neurotoxins, Electrochem. Commun. 2011, 13, 611-614 [IF=5.4]
A novel strip-based disposable amperometric proton sensor that can selectively detect organophosphate neurotoxins (i.e., paraoxon) is described. The detection methodology is based on measuring the current change involved in the assisted proton transfer by a proton selective ligand (e.g., ETH 1778) across a microelliptic hole interface between the aqueous and polyvinylchloride-2-nitrophenyloctylether gel phase. The selective detection of paraoxon is achieved by measuring protons released by the specific hydrolysis of paraoxon with the organophosphorus hydrolase enzyme. A two-step process involving the hydrolysis and proton transfer reaction was characterized using cyclic voltammetry and differential pulse stripping voltammetry. A strip-based sensor fabricated using a simple polydimethylsiloxane (PDMS) mold with the resulting device was found to exhibit a linear response over a wide range of paraoxon concentrations (0.5 μM– 100 μM) present in aqueous samples. In addition to the excellent detection limit and a wide dynamic range, a superb selectivity in the presence of common interfering agents in agricultural samples is achieved.
1. Jeong Hyun Seo, Chang Sup Kim, Byeong Hee Hwang, Hyung Joon Cha*, A functional carbohydrate chip platform for analysis of carbohydrate-protein interaction, Nanotechnology 2010, 21, 215101-215108 [IF=3.5]
A carbohydrate chip based on glass or other transparent surfaces has been suggested as a potential tool for high-throughput analysis of carbohydrate–protein interactions. Here we proposed a facile, efficient, and cost-effective method whereby diverse carbohydrate types are modified in a single step and directly immobilized onto a glass surface, with retention of functional orientation. We modified various types of carbohydrates by reductive amination, in which reducing sugar groups were coupled with 4-(2-aminoethyl)aniline, which has di-amine groups at both ends. The modified carbohydrates were covalently attached to an amino-reactive NHS-activated glass surface by formation of stable amide bonds. This proposed method was applied for efficient construction of a carbohydrate microarray to analyze carbohydrate–protein interactions. The carbohydrate chip prepared using our method can be successfully used in diverse biomimetic studies of carbohydrates, including carbohydrate–biomolecule interactions, and carbohydrate sensor chip or microarray development for diagnosis and screening.