• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.171-173
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• 2003

Suspension culture in serum-free medium is important for the efficient large-scale culture of anchorage-dependent cells that are utilized to produce therapeutic recombinant protein(e.g., insulin, antibody, vaccine) and virus vector for therapeutic gene transfer. We developed a novel method for the suspension culture of anchorage-dependent animal cells in serum-free medium using biodegradable polymer nanospheres in this study. Poly(lactic-co-glycolic acid) (PLGA) polymer nanospheres (433nm in average diameter) were used to the culture of human embryonic kidney 293 cells in serum-free medium in stirred suspension bioreactors. The use of PLGA nanospheres promoted the aggregate formation and cell growth (3.8-fold versus 1.8-fold growth), compared to culture without nanospheres. Adaptation of the anchorage-dependent cells to suspension culture or serum-free medium is time-consuming and costly. In contrast, the culture method developed in our study does not require the adaptation process. This method may be useful for the large-scale suspension culture of various types of anchorage-dependent animal cells in serum-free medium.

• The Plant Pathology Journal
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• v.22 no.1
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• pp.68-74
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• 2006

A new virus with a dsRNA genome was isolated and characterized from the Suhan-:neutari strain (ASI 2504) of Pleurotus ostreatus, which was characterized as long and slightly bent with small caps on the stipe of fruit body. Thirty nm isometric viruses with three dsRNA segments (approximately 2.0, 1.84 and 1.82 kb in sizes) were isolated by ultracentrifugation in sucrose gradients. Western analysis of protein extracted purified viruses with anti-virus polyclonal antibody confirmed that viruses have two specific proteins (36 and 68 kDa). Computer analysis of 2.0 kb segment shows that high. sequence identity with RNA-dependent RNA polymerase (RdRp) of partitiviruses, respectively. When compared to other dsRNA mycoviruses in a phylogenetic analysis, OMDV was most related to Pleurotus ostreatus virus 1.

• KSBB Journal
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• v.32 no.3
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• pp.193-198
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• 2017

Sialylation is important in producing therapeutic proteins such as antibody, cytokine and fusion protein. Thus, enhancement of sialylation is usually performed in CHO cell cultures. ${\alpha}2,6$-Sialyltransferase (ST), which plays a key role in the attachment of ${\alpha}2,6-sialic$ acid, is present in human cells but not in Chinese hamster ovary (CHO) cells. Overexpression of ${\alpha}2,6-ST$ can be used for enhancing the degree of sialylation and achieving human-like glycosylation. In this study, we constructed CHO cells producing human cytotoxic T-lymphocyte antigen4-immunoglobulin (hCTLA4-Ig) as well as ${\alpha}2,6-ST$. Transfected CHO cells were selected using G418 and stable cell line was established. Profiles of viable cell density and hCTLA4-Ig titer in an overexpressed cell line were similar to those of a wild-type cell line. It was confirmed that the total amount of sialic acid was increased and ${\alpha}2,6-sialic$ acid was attached to the terminal residues of N-glycan of hCTLA4-Ig by ESI-LC-MS. Compared to 100% of ${\alpha}2,3-sialic$ acid in wild type cells, 70.9% of total sialylated N-glycans were composed of ${\alpha}2,6-sialic$ acid in transfected cells. In conclusion, overexpression of ${\alpha}2,6-ST$ in CHO cells led to the increase of both the amount of total sialylated N-glycan and the content of ${\alpha}2,6-sialic$ acid, which is more resemble to human-like structure of glycosylation.

• BMB Reports
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• v.33 no.3
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• pp.242-248
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• 2000

Phage display of proteins is a powerful tool for protein engineering since a vast library of sequences can be rapidly screened for a specific property. In this study, we develop da new phage display vector that was derived from a pET-25b(+) vector. The pET-25b(+) was modified in order that the expressed protein would have a T7-tag at the amino terminus and GpS (a major coat protein of M13 phage) at the carboxyl terminus. Another vector without the gp8 gene was also constructed. The newly developed phagemid vectors have several advantageous features. First, it is easy to examine whether or not the target proteins are functional and faithfully transported into the periplasmic space. This feature is due to the fact that recombinant proteins are produced abundantly in the pET system. Second, the T7-tag makes it possible to detect any target proteins that are displayed on the surface of filamentous bacteriophage. To verify the utility of the vector, the clones containing the glutathione S-transferase (GST) gene as a target were examined. The result showed that the GST produced from the recombinant vector was successfully transported into the periplasmic space and had the anticipated enzyme activity. Western blot analysis using a T7-tag antibody also showed the presence of the target protein displayed on the surface of the phage. The phages prepared from the recombinant clones were able to bind to glutathione-Sepharose and then eluted with glutathione. These results showed that the new vectors developed in this study are useful for the phage display of proteins.

• Korean Journal of Plant Tissue Culture
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• v.21 no.6
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• pp.357-362
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• 1994

Chitinases and $\beta$-1,3-glucanases are believed to be important in defending plane against pathogens. Here, we investigated the expression of chitinase(s) in Arabidopsis thaliana cell suspension culture system in response to ethephon (2-chloroethyl phosphonic acid) which produces ethylene or a microbial elicitor, a bacterial pectin-degrading enzyme, $\beta$-1, 4-endopolygalactronic acid Iyase (PGA Iyase), treatment. Chitinase activity was measured either by radio chemical assay using $^3$H-labeled regenerated chitin as substrate or western blot analysis using antibody raised against tobacro chitinase(S). With 1 mg/mL of ethephon or 100 m units/mL of elicitor treatment, maximum levels of activity were reached after 48h. We also investigated distribution of chitinase activity in seedlings, leaves, and root of A. thaliana and found that root have the highest chitinase activity.

• Applied Chemistry for Engineering
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• v.29 no.1
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• pp.97-102
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• 2018

In this work, the surface of gold nanoparticles (AuNPs) was modified with small molecules including mercaptoundecanoic acid (MUA) and L-lysine for the development of highly sensitive lateral flow (LF) sensors. Uniformly sized AuNps were synthesized by a modified Turkevich-Frens method, showing an average size of $16.7{\pm}2.1nm$. Functionalized AuNPs were then characterized by transmission electron microscopy, UV-vis spectroscopy, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy. The stable conjugation of AuNPs and antibodies was obtained at pH 7.07 and the antibody concentration of $10{\mu}g/mL$. The functionalized AuNP-based LF sensor exhibited lower detection limit of 10 ng/mL for hepatitis B surface antigens than that of using the bare AuNP-based LF sensor (100 ng/mL).

• Journal of Microbiology and Biotechnology
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• v.9 no.5
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• pp.541-547
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• 1999

Constructions of a transfer vector and a recombinant baculovirus using the thymidine kinase gene of the Herpes simplex virus type 1 strain F (HSV -1) were carried out. Newly cloned transfer vector, pHcgXIIIB, was constructed by insertion of the glycoprotein gX gene signal peptide sequence of Pseudorabies virus into the baculovirus vector pHcEV-IV. The gX sequence was inserted just downstream from the promoter for the polyhedrin gene of the Hyphantria cunea nuclear polyhedrosis virus (HcNPV). HSV-1 thymidine kinase(tk) gene (1.131 kb) was used as a candidate gene for transferring into the baculovirus expression system. The tk gene was inserted into a BamHI site downstream from the gX sequence-promoter for the polyhedrin gene in the pHcgXIIIB transfer vector and was transferred into the infectious lacZ-HcNPV expression vector. Recombinant virus was isolated and was named gX-TK-HcNPV. The recombinant virus produced a 45 kDa gX-TK fusion protein in Spodoptera frugiperda cells, which was confirmed by Western blot analysis. Microscopic examination of gX-TK-HcNPV-infected cells revealed normal multiplication. Fluorescent antibody staining indicated that the gX-TK fusion protein was present in the cytoplasm. These results indicated that the transfer vector successfully transferred the gX-tk gene into the baculovirus expression system.

• Proceedings of the Korean Institute of Surface Engineering Conference
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• 2012.11a
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• pp.4-4
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• 2012

Proteins enable biology to be viable through molecular interactions (such as in antigen-antibody, ligand-receptor, and drug-target) with

• Journal of Ginseng Research
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• v.41 no.3
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• pp.268-276
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• 2017

Background: UV-B-exposed keratinocytes secrete various paracrine factors. Among these factors, basic fibroblast growth factor (bFGF) stimulates the proliferation of melanocytes. Ginsenosides, the major active compounds of ginseng, are known to have broad pharmacological effects. In this study, we examined the antiproliferative effects of ginsenosides on bFGF-induced melanocyte proliferation. Methods: We investigated the inhibitory effects of Korean Red Ginseng and ginsenosides from Panax ginseng on bFGF-induced proliferation of melan-a melanocytes. Results: When melan-a melanocytes were treated with UV-B-irradiated SP-1 keratinocytes media, cell proliferation increased. This increased proliferation of melanocytes decreased with a neutralizing anti-bFGF antibody. To elucidate the effects of ginsenosides on melanocyte proliferation induced by bFGF, we tested 15 types of ginsenoside compounds. Among them, Rh3, Rh1, F1, and CK demonstrated antiproliferative effects on bFGF-induced melanocyte proliferation after 72 h of treatment. bFGF stimulated cell proliferation via extracellular signal-regulated kinase (ERK) activation in various cell types. Western blot analysis found bFGF-induced ERK phosphorylation in melan-a. Treatment with Rh3 inhibited bFGF-induced maximum ERK phosphorylation and F1-delayed maximum ERK phosphorylation, whereas Rh1 and CK had no detectable effects. In addition, cotreatment with Rh3 and F1 significantly suppressed bFGF-induced ERK phosphorylation. Western blot analysis found that bFGF increased microphthalmia-associated transcription factor (MITF) protein levels in melan-a. Treatment with Rh3 or F1 had no detectable effects, whereas cotreatment with Rh3 and F1 inhibited bFGF-induced MITF expression levels more strongly than a single treatment. Conclusion: In summary, we found that ginsenosides Rh3 and F1 have a synergistic antiproliferative effect on bFGF-induced melan-a melanocyte proliferation via the inhibition of ERK-mediated upregulation of MITF.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.587-594
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• 2000

Monoclonal antibodies against glutamate dehydrogenase (GDH) from Sulfolobus solfataricus were produced and characterized using epitope mapping and biosensor technology, Five monoclonal antibodies raised against S. solfataricus GDH were each identified as a single protein band that comigrated with purified S. solfataricus GDH on the SDS-polyacrylamide gel electrophoresis and immunoblot. Epitope mapping analysis showed that only one subgroup among the antibodies tested recognized the same peptide fragments of GDH. Using the anti-S. solfataricus GDH antibodies as probes, the cross-reactivities of GDHs from various sources were investigated and it was found that the mammalian GDH is not immunologically related to S. solfataricus GDH. The structural differences between the microbial and mammalian GDHs were further investigated using biosensor technology (Pharmacia BIAcore) and monoclonal antibodies against S. solfataricus and bovine brain. The binding affinity of S. solfataricus glutamate dehydrogenase anti-S. solfataricus for GDH ($K_D$=11 nM) was much tighter than that of anti-bovine for GDH ($K_D$=450 nM). These results, together with the epitope mapping analysis, suggest that there may be structural differences between the two GDH species, in addition to their different biochemical properties.