• Proceedings of the Korean Vacuum Society Conference
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• 2013.02a
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• pp.84-84
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• 2013

FcBP consisting of 13 amino acids specifically binds to Immunoglobulin G Fc domain. Initially, we utilized this peptide for preparation of antibody chip as a PEG composite for enhanced solubility. After then, the peptide conjugate was immobilized on agarose resin, resulting in highly efficient affinity column for antibody purification. The efficiency was comparable to commercial Protein A column. Recently, this peptide was conjugated with cell penetratingpeptide (CPP) on a backbone of GFP, affording antibody transducer, which carries antibody into live cells by simple mixing of antibody and the transducer in cell culture media. Antibody transduction into cells was monitored by live cell imaging. More recently, the FcBP was fused to ferritin cage, which consists of 24 ferritin protein molecules. The FcBP-ferritin cage showed greatly increased binding affinity to human IgG. Its binding was analyzed by QCM and SPR analysis. Finally, it was selectively delivered by Herceptin to SKBR3, a breast cancer cell, over MCF10A, non-tumorigenic cells.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2018.10a
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• pp.444-447
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• 2018

Baculovirus expression systems have many known advantages including fast and cost-effective methods to generate large amounts of recombinant proteins in comparison to bacterial expression systems, particularly those requiring complex post-translational modifications. Especially, recombinant baculoviruses can transfer their vectors and express their recombinant proteins in a wide range of mammalian cell types. In this study, baculoviral vectors which were reconstructed from pcDNA3.1 vector, were recombined with cytomegalovirus (CMV) promoter,uroplakin II promoter, polyhedron promoter, vesicular stomatitis virus G (VSVG), enhanced green fluorescent protein (EGFP), and protein transduction domain (PTD). These recombinant vectors were infected with various cells and cell lines. The baculovirus vector thus developed was analyzed by comparing the metastasis and expression of the recombinant genes with conventional vectors. These results suggest that the baculovirus vector has higher efficiency in metastasis and expression than the control vector.

• BMB Reports
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• v.46 no.11
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• pp.561-566
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• 2013

We examined the ways in which fenobam could promote not only the transduction of PEP-1-FK506BP into cells and tissues but also the neuroprotective effect of PEP-1-FK506BP against ischemic damage. Fenobam strongly enhanced the protective effect of PEP-1-FK506BP against $H_2O_2$-induced toxicity and DNA fragmentation in C6 cells. In addition, combinational treatment of fenobam with PEP-1-FK506BP significantly inhibited the activation of Akt and MAPK induced by $H_2O_2$, compared to treatment with PEP-1-FK506BP alone. Interestingly, our results showed that fenobam significantly increased the transduction of PEP-1-FK506BP into both C6 cells and the hippocampus of gerbil brains. Subsequently, a transient ischemic gerbil model study demonstrated that fenobam pretreatment led to the increased neuroprotection of PEP-1-FK506BP in the CA1 region of the hippocampus. Therefore, these results suggest that fenobam can be a useful agent to enhance the transduction of therapeutic PEP-1-fusion proteins into cells and tissues, thereby promoting their neuroprotective effects.

• Proceedings of the Korean Vacuum Society Conference
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• 2013.02a
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• pp.110-111
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• 2013

FcBP consisting of 13 amino acids specifically binds to Immunoglobulin G Fc domain. Initially, we utilized this peptide for preparation of antibody chip as a PEG composite for enhanced solubility. After then, the peptide conjugate was immobilized on agarose resin, resulting in highly efficient affinity column for antibody purification. The efficiency was comparable to commercial Protein A column. Recently, this peptide was conjugated with cell penetrating peptide (CPP) on a backbone of GFP, affording antibody transducer, which carries antibody into live cells by simple mixing of antibody and the transducer in cell culture media. Antibody transduction into cells was monitored by live cell imaging. More recently, the FcBP was fused to ferritin cage, which consists of 24 ferritin protein molecules. The FcBP-ferritin cage showed greatly increased binding affinity to human IgG. Its binding was analyzed by QCM and SPR analysis. Finally, it was selectively delivered by Herceptin to SKBR3, a breast cancer cell, over MCF10A, non-tumorigenic cells (Fig. 1). Fig. 1. Fluorescent microscopic images of SKBR3 breast cancer cells (A~C) and MCF10A breast cells (D~F) treated with Cy3-trastuzumab/fFcBP-Pf_Fn complexes. Trastuzumab and FcBP-Pf_Fn, which were labeled with Cy3 (Cy3-trastuzumab) and fluorescein (fFcBP-Pf_Fn), respectively, selectively targeted SKBR3 over MCF10A.

• Korean Journal of Microbiology
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• v.47 no.1
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• pp.1-6
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• 2011

Inefficiency of in vivo gene transfer using currently available vectors reflects a major hurdle in cancer gene therapy. Both viral and non-viral approaches have been described to improve gene transfer efficiency but suffer from a number of limitations. Here we tested an adenovirus carrying the small peptide ligand derived from heregulin${\beta}$ EGF-like domain onto fiber, the adenoviral capsid protein, to deliver transgene to ovarian cancer cells which overexpress ErbB, the cognate receptors for heregulin. The attachement of 53 amino acids to fiber didn't affect on the fiber's trimer structure that is critical for the viral entry to cells. The fiber-modified adenovirus can mediate entry and expression of a ${\beta}$-galactosidase into cancer cells in an increased efficiency compared the unmodified adenovirus. Particularly, the gene transfer efficiency was improved up to 5 times in OVCAR3 cells, an ovarian cancer cell line. Such transduction systems hold promise for delivering genes to ErbB receptor overexpressing cancer cells, and could be used for future cancer gene therapy.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.582-586
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• 2003

There has been interest in developing gene therapy based on naked plasmid DNA for treating serum protein deficiencies and human erythropoietin (hEPO) is one of the candidate for gene therapy being Investigated most enthusiastically. We constructed novel plasmid DNA vectors pVAC-hEPOI/II/III which contain one, two, three hEPO gene(s) respectively for producing high level expression and secretion of hEPO in vitro and in vivo. NIH3T3 and COS7 cells were transfected transiently with these vectors and increase in hEPO expression in medium reached 2-5 fold in comparison with pSecTagB-hEPO. Intra muscular administrations of pVAC-hEPOI/II/III vectors into mice resulted in high level secretion of hEPO in the serum and corresponding increases in hematocrit level. In conclusion the transduction efficiency of naked plasmid vectors is one of the critical factors of a gene delivery system and these novel plasmid vectors will contribute to various gene therapy based on naked plasmid DNA.

• KSBB Journal
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• v.24 no.1
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• pp.80-88
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• 2009

Human prostatic acid phosphatase (PAP), with comprehensive homology to glandular kallikrein, are representative serum biomarkers of prostate cancer. Dendritic cell (DC), which is the potent antigen-presenting cells(APC) in the immune system, can induce strong T cell responses against viruses, microbial pathogens, and tumors. Therefore, the immunization using DC loaded with tumor-associated antigens is a powerful method for inducing anti-tumor immunity. The CTP (Cytoplasmic Transduction Peptide) technology developed by Creagene which can transport attached bio-polymers like nucleic acids or proteins into the cell with high permeation efficiency. As the active forms of PAP can mediate apoptotic processing, we used multimer forms of PAP as an inactive form for antigen pulsing of DCs. In this study, multimeric forms of CTP-rhPAP was obtained according to the advanced purification process and subsequently confirmed by gel filtration chromatography, western blot and Dynamic Light Scattering. Therefore, CTP-conjugated PA multimers transduced into the cytoplasm were efficiently presented on the cell surface without any harm effect on cells via MHC class I molecules and result in induction of a large number of effector cell.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2019.05a
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• pp.533-536
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• 2019

Recombinant baculoviruses are widely used to express heterologous genes in cultured insect cells. Recombinant baculoviruses can serve as gene-transfer vectors for expression of recombinant proteins in a wide range of mammalian cell types. Baculovirus system has significant benefits in view of safety, large-scale, and high level of gene expression. In this study, baculoviral vectors which were reconstructed from pOPINEneo-3C-GFP vector, were recombined with cytomegalovirus (CMV) promoter, green fluorescent protein (GFP), and p53 with NcoI and XhoI. These recombinant vectors were infected with various cells and cell lines. The baculovirus vector thus developed was analyzed by comparing the metastasis and expression of the recombinant genes with conventional vectors. These results suggest that the baculovirus vector has higher efficiency in metastasis and expression than the control vector. This work was supported by a grant from Mid-Career Researcher Program(NRF-2016R1A2B4016552) through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning(MSIP).

• Nuclear Medicine and Molecular Imaging
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• v.42 no.5
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• pp.394-400
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• 2008

Purpose: Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirus-mediated delivery systems has not been reported. Here, we evaluated the human sodium iodide symporter (hNIS) gene expression in rat mesenchymal stem cell (rMSC) transduced by lentivirus or adenovirus, and compared the hNIS expression quantitatively between the two delivery systems. Materials and Methods: Lentiviral-mediated hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning hNIS gene into pLenti6/UbC/V5-DEST (Invitrogen) to obtain pLenti-hNIS, transducing rMSC with the pLenti-hNIS, and selecting with blasticidin for 3 weeks. Recombinant adenovirus expressing hNIS gene (Rad-hNIS) was produced by homologous recombination and transduction efficiency of Rad-hNIS into rMSC evaluated by Rad-GFP was $19.1{\pm}4.7%$, $54.0{\pm}6.4%$, $85.7{\pm}8.7%$, and $98.4{\pm}1.3%$ at MOI 1, 5, 20, and 100, respectively. The hNIS expressions in lenti-hNIS-rMSC or adeno-hNIS-rMSC were assessed by immunocytochemistry, western blot, and 1-125 uptake. Results: Immunocytochemistry and western blot analyses revealed that hNIS expressions in lenti-hNIS-rMSC were greater than those in adeno-hNIS-rMSC at MOI 20 but lower than at MOI 50. However in vitro 1-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC ($29,704{\pm}6,659\; picomole/10^6\;cells$) was greater than that in adeno-hNIS-rMSC at MOI 100 ($6,168{\pm}2,134\;picomole/10^6\;cells$). Conclusion: Despite lower amount of expressed protein, hNIS function in rMSC was greater by lentivirus than by adenovirus mediated expression. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative vector efficiency for transgene expression.

• Proceedings of the Microbiological Society of Korea Conference
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• 2007.05a
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• pp.120-122
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• 2007

All living organisms use numerous signal-transduction pathways to sense and respond to their environments and thereby survive and proliferate in a range of biological niches. Molecular dissection of these signalling networks has increased our understanding of these communication processes and provides a platform for therapeutic intervention when these pathways malfunction in disease states, including infection. Owing to the expanding availability of sequenced genomes, a wealth of genetic and molecular tools and the conservation of signalling networks, members of the fungal kingdom serve as excellent model systems for more complex, multicellular organisms. Here, we employed Cryptococcus neoformans as a model system to understand how fungal-signalling circuits operate at the molecular level to sense and respond to a plethora of environmental stresses, including osmoticshock, UV, high temperature, oxidative stress and toxic drugs/metabolites. The stress-activated p38/Hog1 MAPK pathway is structurally conserved in many organisms as diverse as yeast and mammals, but its regulation is uniquely specialized in a majority of clinical Cryptococcus neoformans serotype A and D strains to control differentiation and virulence factor regulation. C. neoformans Hog1 MAPK is controlled by Pbs2 MAPK kinase (MAPKK). The Pbs2-Hog1 MAPK cascade is controlled by the fungal "two-component" system that is composed of a response regulator, Ssk1, and multiple sensor kinases, including two-component.like (Tco) 1 and Tco2. Tco1 and Tco2 play shared and distinct roles in stress responses and drug sensitivity through the Hog1 MAPK system. Furthermore, each sensor kinase mediates unique cellular functions for virulence and morphological differentiation. We also identified and characterized the Ssk2 MAPKKK upstream of the MAPKK Pbs2 and the MAPK Hog1 in C. neoformans. The SSK2 gene was identified as a potential component responsible for differential Hog1 regulation between the serotype D sibling f1 strains B3501 and B3502 through comparative analysis of their meiotic map with the meiotic segregation of Hog1-dependent sensitivity to the fungicide fludioxonil. Ssk2 is the only polymorphic component in the Hog1 MAPK module, including two coding sequence changes between the SSK2 alleles in B3501 and B3502 strains. To further support this finding, the SSK2 allele exchange completely swapped Hog1-related phenotypes between B3501 and B3502 strains. In the serotype A strain H99, disruption of the SSK2 gene dramatically enhanced capsule biosynthesis and mating efficiency, similar to pbs2 and hog1 mutations. Furthermore, ssk2, pbs2, and hog1 mutants are all hypersensitive to a variety of stresses and completely resistant to fludioxonil. Taken together, these findings indicate that Ssk2 is the critical interface protein connecting the two-component system and the Pbs2-Hog1 pathway in C. neoformans.