• Bulletin of the Korean Chemical Society
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• v.25 no.9
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• pp.1366-1370
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• 2004

Controlled pore glass bead was modified with bovine serum albumin (BSA), and glutathione (GSH) was immobilized through three kinds of linkers on top of BSA. Bis(3-sulfo-N-hydroxysuccinimide suberate) sodium salt $(BS^3)$, N-hydroxysuccinimide 3-(2-pyridyldithio)propionate (SPDP), or N-hydroxysuccinimide 4-maleimidobutyrate (GMBS) was introduced into the BSA-bound matrix. Subsequently, GSH was immobilized by addition of thiol side chain into the maleimido moiety, replacing a disulfide group, or formation of an amide group upon releasing 3-sulfo-N-hydroxysuccimide group. It was observed that conjugation methodology played a critical role for activity of the immobilized GSH. SDS-PAGE chromatogram showed that the matrix of glutathione immobilized on BSA through GMBS manifested high selectivity towards glutathione-S-transferase (GST) in cell lysate.

• Biomedical Science Letters
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• v.9 no.3
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• pp.111-121
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• 2003

Recombinant single chain Fv (scFv) antibodies offer many advantages over mouse monoclonal antibodies such as faster clearance from blood, improved tumor localization, reduced human anti-mouse antibody (HAMA) response, and the availability to manipulate the scFv through genetic approaches. The recombinant phage display was constructed using lym-l hybridoma cells as a source of genetic starting material. mRNA was isolated from the corresponding antibodies hybridoma cells. VH and VL cDNA were amplified with RT-PCR and linked with ScFv by linker DNA to form ScFv DNA, which then were inserted into phagemid pCANTAB5E. The phage of positive clones selected with tube containing raji lymphoma cell and infected by competent E. coli HB2151 to express soluble scFv. The scFv lym-l was secreted into the cytosol and culture supernatant and shown to be of expected size (approximately 32 kDa) by western blot. An active scFv lym-l could be produced in E. coli with soluble form and high yield from hybridoma cell line, using phage display system. Immunoreactivity indicated that scFv lym1 showed a potential biding affinity against the raji lymphoma cell as its parental antibody (intact lym-l Ab).

• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.376-380
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• 1999

Constructs, encoding a single-chain variable fragment of a catalytic antibody 4f4f (scFv-4f4f) with glycosidase activity, were made by combining the coding sequences for the heavy and light chain variable domains with a sequence encoding a linker (GGGGS). Using three different plasmid systems, single-chain antibodies were expressed separately in Escherichia coli, demonstrating significant differences in the expression level and amounts in soluble form of the recombinant protein. The protein expression from pET3a-scFv-4f4f was up to 20% of the total soluble proteins and, more importantly, the proteins were mostly found in a soluble form. An SDS-PAGE analysis of the purified single-chain proteins, yielding higher than 5mg from a 1-1 culture, showed a single band corresponding to its molecular weight of 29,100. A preliminary study shows that the expressed scFv-4f4f is catalytically active. The catalytic parameters for the hydrolysis of p-nitrophenyl-$\beta$-D-glucopyranoside by scFv-4f4f are being investigated.

• Journal of the Korean Electrochemical Society
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• v.13 no.2
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• pp.145-152
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• 2010

The gel polymer electrolyte(GPE) were prepared by in-situ thermal cross-linking reaction of homogeneous precursor solution of perfluorinated phosphate-based cross-linker and liquid electrolyte. Ionic conductivities and electrochemical properties of the prepared gel polymer electrolyte with the various contents of liquid electrolytes and perfluorinated organophosphate-based cross-linker were examined. The stable gel polymer electrolyte was obtained up to 97 wt% of the liquid electrolyte. Ionic conductivity and electrochemical properties of the gel polymer electrolytes with the various chain length of perfluorinated ethylene oxide and different content of liquid electrolytes were examined. The maximum ionic conductivity of liquid electrolyte was measured to be $1.02\;{\times}\;10^{-2}\;S/cm$ at $30^{\circ}C$ using the cross-linker($PFT_nGA$). The electrochemical stability of the gel polymer electrolyte was extended to 4.5 V. The electrochemical performances of test cells composed of the resulting gel polymer electrolyte were also studied to evaluate the applicability on the lithium polymer batteries. The test cell carried a discharge capacity of 136.11mAh/g at 0.1C. The discharge capacity was measured to be 91% at 2C rate. The discharge capacity decreased with increase of discharge rate which was due to the polarization. After 500th charge/discharge cycles, the capacity of battery decreased to be 70% of the initial capacity.

• Toxicological Research
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• v.17
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• pp.109-115
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• 2001

Abrus agglutinin was purified from the kernels of Abrus precatorius by Sepharose 4B affinity column chromatography followed by Sephadex G-100 gel filtration column chromatography. About 1.25 g of abrus agglutinin was obtained from 1 kg of the kernels. The LD$_{50}$ of abrus agglutinin is 5 mg/kg of body weight, which is less toxic than that of abrin, 20$\mu\textrm{g}$/kg body weight. The amino acid sequence of abrus agglutinin was determined by protein sequencing techniques and deduced from the nucleotide sequence of a cDNA clone encoding full length of abrus agglutinin. There are 258 residues, 2 residues and 267 residues in the A-chain, the linker peptide and the B-chain of abrus agglutinin, respectively. Abrus agglutinin had high homology to abrin-a (77.8%). The 13 amino acid residues involved in catalytic function, which are highly conserved among abrin and ricin, were also conserved within abrus agglutinin. The protein synthesis inhibitory activity of abrus agglutinin ($IC_{50}$/ = 3.5 nM) was weaker than that of abrin-a (0.05 nM). By molecular modeling followed by site-directed mutagenesis showed that Pro199 of abrus agglutinin A-chain located in amphipathic helix H and corresponding to Asn200 of abrin A-chain, can induce bending of helix H. This bending would presumably affect the binding of abrus agglutinin A-chain to its target sequence GpApGpAp, in the tetraloop structure of 285 r-RNA subunit and this could be one of major factors contributing to the relatively weak protein synthesis inhibitory activity and toxicity of abrus agglutinin.n.

• Journal of the Korean Ceramic Society
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• v.59
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• pp.527-535
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• 2020

This study reports the preparation of the Candida antarctica lipase B (CalB) enzyme immobilization on silica-coated magnetic nanoparticles (Si-MNPs@CalB) using various cross-linkers and demonstration of rapid catalytic hydrolysis of p-nitrophenyl alkyl esters. CalB enzymes were coupled with different cross-linker silanes on the Si-MNPs surface. Among these cross-linkers, Cl-functionalized silane was better at immobilization of CalB than the others. Catalytic hydrolysis of p -nitrophenyl alkyl esters was demonstrated against Si-MNPs@CalB as a function of the length of alkyl chain (C₄, C₈, C₁₂, and C₁₆). From the Michaelis-Menten equation and Lineweaver-Burk plots, various enzyme kinetic parameters (i.e., K_m, V_max, and K_cat) were calculated. Catalytic hydrolysis was faster in shorter alkyl chain of p-nitrophenyl alkyl esters with Si-MNPs@CalB in the order C₄>>C₈>C₁₂>>C₁₆. Furthermore, the reusability and optimum catalytic activity of Si-MNPs@ CalB were evaluated as a function of the number of reuses and with different pH values.

• IMMUNE NETWORK
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• v.12 no.1
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• pp.33-39
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• 2012

Background: Therapeutic approaches using monoclonal antibodies (mAbs) against complement regulatory proteins (CRPs:i.e.,CD46,CD55 and CD59) have been reported for adjuvant cancer therapy. In this study, we generated a recombinant 1E8 single-chain anti-CD59 antibody (scFv-Fc) and tested anti-cancer effect.by using complement dependent cytotoxicity (CDC). Methods: We isolated mRNA from 1E8 hybridoma cells and amplified the variable regions of the heavy chain (VH) and light chain (VL) genes using reversetranscriptase polymerase chain reaction (RT-PCR). Using a linker, the amplified sequences for the heavy and light chains were each connected to the sequence for a single polypeptide chain that was designed to be expressed. The VL and VH fragments were cloned into the pOptiVEC-TOPO vector that contained the human CH2-CH3 fragment. Then, 293T cells were transfected with the 1E8 single-chain Fv-Fc (scFv-Fc) constructs. CD59 expression was evaluated in the prostate cancer cell lines using flow cytometry. The enhancement of CDC effect by mouse 1E8 and 1E8 scFv-Fc were evaluated using a cytotoxicity assay. Results: The scFv-Fc constructs were expressed by the transfected 293T cells and secreted into the culture medium. The immunoreactivity of the secreted scFv-Fc construct was similar to that of the mouse 1E8 for CCRF-CEM cells. The molecular masses of 1E8 scFv-Fc were about 120 kDa and 55 kDa under reducing and non-reducing conditions, respectively. The DNA sequence of 1E8 scFv-Fc was obtained and presented. CD59 was highly expressed by the prostate cancer cell line. The recombinant 1E8 scFv-Fc mAb revealed significantly enhanced CDC effect similar with mouse 1E8 for prostate cancer cells. Conclusion: A 1E8 scFv-Fc construct for adjuvant cancer therapy was developed.

• Korean Journal of Poultry Science
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• v.38 no.4
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• pp.323-330
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• 2011

The chicken monoclonal antibody (mAb), 13C8, reacts with sporozoite antigens of Eimeria spp. which causes avian coccidiosis. Since this mAb was produced at low amount due to genetic instability of chicken hybridoma, a recombinant 13C8 single-chain Fv (ScFv) antibody was constructed by amplification of the variable domain of heavy (VH) and light chain (VL) genes of antibody derived from chicken hybridoma. The constructed 13C8 ScFv was successfully expressed in E. coli and purified as a soluble form. In ELISA analysis, this recombinant 13C8 ScFv antibody showed antigen binding activity as the original mAb. In addition, nucleotide sequence comparison of 13C8 gene to the germline chicken VL and VH genes suggested that the gene conversion with $V{\lambda}$ and VH pseudogenes might contribute to the diversification of VL and VH genes in chickens.

• Journal of Life Science
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• v.30 no.11
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• pp.1012-1020
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• 2020

Remicade is a therapeutic biosimilar natural antibody in which the mouse variable domain has been linked to the human constant domain. It is a chimeric monoclonal antibody specific to tumor necrosis factor-alpha (TNF-α) and has been developed for the treatment of rheumatoid arthritis. To investigate the biological activity of the Remicade antibody, we carried out a bioinformatics study using a protein data bank to characterize the TNF-α antigen binding mechanism of the Remicade natural antibody. Because the production of the Remicade antibody is often limited by genetic instability of the natural antibody-producing cell, we generated a Remicade single-chain variable domain fragment antibody (Remicade) in which a heavy chain variable domain (VH) is joined with a light chain variable domain (VL) by a polypeptide linker. Furthermore, Remicade was fused to a leucine zipper (RemicadeScZip) for higher production and higher antigen-binding activity than Remicade. The Remicade and Remicade ScZip were expressed in Escherichia coli and purified by a Ni⁺-NTA-agarose column. As expected, the purified proteins had migrated as 28.80 kDa and 33.96 kDa in sodium dodecyl sulfate-polyacrylamide electrophoresis. The TNF-α antigen binding activity of Remicade was not observed by ELISA and western blot. In contrast, RemicadeScZip showed antigen-binding activity. Additional bio-layer interferometry analysis confirmed the antigen-binding activity of RemicadeScZip, suggesting that the leucine zipper stabilized the folding of RemicadeScZip in a denatured condition and improved the TNF-α antigenbinding activity.

• Journal of Microbiology and Biotechnology
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• v.21 no.5
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• pp.529-535
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• 2011

Single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulin, connected with a short linker peptide of 10 to about 20 amino acids. In this study, the scFv of a monoclonal antibody against the third domain of human CD4 was cloned from OKT4 hybridoma cells using the phage display technique and produced in E. coli. The expression, production, and purification of anti-CD4 scFv were tested using SDS-PAGE and Western blot, and the specificity of anti-CD4 scFv was examined using ELISA. A 31 kDa recombinant anti-CD4 scFv was expressed and produced in bacteria, which was confirmed by SDS-PAGE and Western blot assays. Sequence analysis proved the ScFv structure of the construct. It was able to bind to CD4 in quality ELISA assay. The canonical structure of anti-CD4 scFv antibody was obtained using the SWISS_MODEL bioinformatics tool for comparing with the scFv general structure. To the best of our knowledge, this is the first report for generating scFv against human CD4 antigen. Engineered anti-CD4 scFv could be used in immunological studies, including fluorochrome conjugation, bispecific antibody production, bifunctional protein synthesis, and other genetic engineering manipulations. Since the binding site of our product is domain 3 (D3) of the CD4 molecule and different from the CD4 immunological main domain, including D1 and D2, further studies are needed to evaluate the anti-CD4 scFv potential for diagnostic and therapeutic applications.