• BMB Reports
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• v.52 no.5
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• pp.324-329
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• 2019

Recent progress in cellular reprogramming technology and lineage-specific cell differentiation has provided great opportunities for translational research. Because virus-based gene delivery is not a practical reprogramming protocol, protein-based reprogramming has been receiving attention as a safe way to generate reprogrammed cells. However, the poor efficiency of the cellular uptake of reprogramming proteins is still a major obstacle. Here, we reported key factors which improve the cellular uptake of these proteins. Purified red fluorescent proteins fused with 9xLysine (dsRED-9K) as a cell penetrating peptide were efficiently delivered into the diverse primary cells. Protein delivery was improved by the addition of amodiaquine. Furthermore, purified dsRED-9K was able to penetrate all cell lineages derived from mouse embryonic stem cells efficiently. Our data may provide important insights into the design of protein-based reprogramming or differentiation protocols.

• Journal of Microbiology and Biotechnology
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• v.25 no.2
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• pp.268-273
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• 2015

The fluorescent-antibody-to-membrane-antigen (FAMA) test is regarded as the "gold standard" to detect protective antibodies to varicella-zoster virus (VZV) because of its high sensitivity and specificity. Because the classic FAMA test uses an infectious virus for detection of antibodies to VZV, it is labor-intensive, and also requires special equipment for handling the virus. For this reason, we attempted to develop a simple and safe FAMA assay. Because VZV glycoprotein E (gE) is one of the major VZV glycoproteins, we used the gE protein for the FAMA test (gE FAMA). Here, we demonstrate that overexpression of gE in HEK293T cells can be used to measure antibodies in human serum, and that gE FAMA titers are closely correlated with gpEIA ELISA data. These results indicate that our gE FAMA test has the potential to measure antibodies to VZV.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.208-211
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• 2000

The recombinant fluorescent chinese hamster ovary (CHO) cell line was developed and optimized through this study for biomonitoring system. This cell line, called KFC-A10, contains recombinant plasmid(pKCFG) constructed in this study for detecting toxic conditions (Mitomicyn C, EDCs, ${\gamma}-ray$, etc.). It is known that c-Fos is involved in proliferation and differentiation of the signal transduction and overexpression of this gene can lead cell to death under the toxic conditions including apoptosis status. Therefore, pKCFG which has the c-fosSRE::GFP is induced by toxic chemicals, especially DNA damage agents and apoptotic chemicals, and produces green fluorescence protein(GFP) under these toxic conditions. Through the characterization of KFC-A10 using fluorescent assays of GFP, it was shown that KFC-A10 cell line had a manifest GFP expression pattern due to various toxicants especially mitomycin C, ${\gamma}-ray$ and bisphenol A. Therefore this study proved the possibility of using GFP as a reporter for detecting various toxicants

• Molecules and Cells
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• v.27 no.4
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• pp.391-396
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• 2009

Image-based, high-content screening assays demand solutions for image segmentation and cellular compartment encoding to track critical events - for example those reported by GFP fusions within mitosis, signalling pathways and protein translocations. To meet this need, a series of nuclear/cytoplasmic discriminating probes have been developed: DRAQ5$^{TM}$ and CyTRAK Orange$^{TM}$. These are spectrally compatible with GFP reporters offering new solutions in imaging and cytometry. At their most fundamental they provide a convenient fluorescent emission signature which is spectrally separated from the commonly used reporter proteins (e.g. eGFP, YFP, mRFP) and fluorescent tags such as Alexafluor 488, fluorescein and Cy2. Additionally, they do not excite in the UV and thus avoid the complications of compound UV-autofluorescence in drug discovery whilst limiting the impact of background sample autofluorescence. They provide a convenient means of stoichiometrically labelling cell nuclei in live cells without the aid of DMSO and can equally be used for fixed cells. Further developments have permitted the simultaneous and differential labelling of both nuclear and cytoplasmic compartments in live and fixed cells to clearly render the precise location of cell boundaries which may be beneficial for quantitative expression measurements, cell-cell interactions and most recently compound in vitro toxicology testing.

• Journal of Microbiology and Biotechnology
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• v.24 no.4
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• pp.568-576
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• 2014

TIM-1 (also known as KIM-1 and HAVcr-1) is a type I transmembrane glycoprotein member of the TIM family that may play important roles in innate and adaptive immune responses. The overexpression of proteins associated with membrane proteins is a major obstacle to overcome in studies of membrane protein structures and functions. In this study, we successfully coupled the overexpression of the TIM-1 protein with a C-terminal enhanced green fluorescent protein (GFP) tag in Escherichia coli. To the best of our knowledge, this report is the first to describe the overexpression of human TIM-1 in E. coli. The purified TIM-1-EGFP fusion protein recognized and bound directly to apoptotic cells and did not to bind to viable cells. Furthermore, we confirmed that the interactions of TIM-1-EGFP with apoptotic cells were blocked by TIM-1-Fc fusion proteins. This fusion protein represents a readily obtainable source of biologically active TIM-1 that may prove useful in future studies of human TIM-1.

• Journal of Sericultural and Entomological Science
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• v.52 no.1
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• pp.25-32
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• 2014

We produced the transgenic silkworm that expressed the enhanced blue fluorescent protein (EBFP) in the cocoon of silkworms. The EBFP fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, was designed to be secreted into the lumen of the posterior silk glands. The expression of the EBFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the $3{\times}P3$-driven DsRed2 cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. A mixture of the donor and helper vector was micro-injected into 300 eggs of silkworms, Baegokjam. We obtained 5 broods. The cocoon displayed blue fluorescence, proving that the fusion protein was present in the cocoon. Also, the presence of fusion proteins in cocoons was demonstrated by SDS-PAGE and western blot analysis. Accordingly, we suggest that the EBFP fluorescence silk will enable the production of the silk-based biomaterials.

• BMB Reports
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• v.51 no.10
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• pp.526-531
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• 2018

Protein-Protein Interactions (PPIs) play essential roles in diverse biological processes and their misregulations are associated with a wide range of diseases. Especially, the growing attention to PPIs as a new class of therapeutic target is increasing the need for an efficient method of cell-based PPI analysis. Thus, we newly developed a robust PPI assay (SeePPI) based on the co-translocation of interacting proteins to the discrete subcellular compartment 'processing body' (p-body) inside living cells, enabling a facile analysis of PPI by the enriched fluorescent signal. The feasibility and strength of SeePPI (${\underline{S}}ignal$ ${\underline{e}}nhancement$ ${\underline{e}}xclusively$ on ${\underline{P}}-body$ for ${\underline{P}}rotein-protein$ ${\underline{I}}nteraction$) assay was firmly demonstrated with FKBP12/FRB interaction induced by rapamycin within seconds in real-time analysis of living cells, indicating its recapitulation of physiological PPI dynamics. In addition, we applied p53/MDM2 interaction and its dissociation by Nutlin-3 to SeePPI assay and further confirmed that SeePPI was quantitative and well reflected the endogenous PPI. Our SeePPI assay will provide another useful tool to achieve an efficient analysis of PPIs and their modulators in cells.

• Korean Journal of Optics and Photonics
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• v.35 no.1
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• pp.30-34
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• 2024

We report a purely protein-based platform for green fluorescence by mixing silk protein with green fluorescence protein, and also report its enhancement by the incorporation of TiO₂ nanoparticles. The TiO₂ nanoparticles employed have diameters of 100 and 300 nm, with a significant increase in fluorescence (by a factor of 7.5) observed when introducing 300-nm TiO₂ nanoparticles. Furthermore, an increase in particle distribution density is found to enhance fluorescence amplification. These research findings suggest that protein-based fluorescent films can be enhanced by the characteristics of nanoparticles, opening up new possibilities in the fields of optics and fluorescence applications.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.754-758
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• 2008

Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied to Monascus ruber. The optimum cocultivation time was 84 h with an efficiency of 900 to 1,000 transformants when $1{\times}10^6$ spores were used with the same volume of bacteria. The stability of transform ants was over 98% after five generations. When M. ruber was transformed with A. tumefaciens YL-63 containing the green fluorescent protein gene (egfp), the green fluorescent signal was observed throughout hyphae, confirming expression of the gene. This efficient transformation and expression system of M. ruber by ATMT will facilitate the study of this fungus at a molecular genetic level.

• Journal of Microbiology and Biotechnology
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• v.18 no.3
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• pp.472-476
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• 2008

Two isoforms of human CD99 have been identified, but only heterotypic interaction between the isomers was recently demonstrated. In this study, we performed bimolecular fluorescence complementation analysis to further characterize the interaction in vivo. Upon transiently transfecting plasmids expressing either of the two isoforms fused with yellow fluorescent protein (YFP) fragments, all the YFP-tagged CD99 molecules were properly localized on cell surfaces, and formed fluorescent dimers. Interestingly, however, unlike the previous report, the homodimers formed as efficiently as the heterodimer via their extracellular domains, implying its distinct regulatory role through modulating the complex profile.