• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.408-411
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• 2005

The green fluorescent protein (GFP) from the jellyfish aequorea and its fluorescent homologs from Anthozoa corals have become invaluable tools for imaging of cells and tissues. In this study various fluorescent protein such as green fluorescent protein (GFP), yellow fluorescent protein (YFP) and red fluorescent protein (RFP) have been expressed in Escherichia coli. Growth of recombinant cells and production of fluorescent proteins were investigated in shake flasks. Some characteristics of fluorescent proteins was also studied.

• BMB Reports
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• v.35 no.6
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• pp.562-567
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• 2002

In order to find the cellular interaction factors of the Heliothis armigera nuclear polyhedrosis virus capsid protein VP39, a Heliothis armigera cell cDNA library was constructed. Then VP39 was used as bait. The host actin gene was isolated from the cDNA library with the yeast two-hybrid system. This demonstrated that VP39 could interact with its host actin in yeast. In order to corroborate this interaction in vivo, the vp39 gene was fused with the green fluorescent protein gene in plasmid pEGFP39. The fusion protein was expressed in the Hz-AM1 cells under the control of the Autographa californica multiple nucleopolyhedrovirus immediate early gene promoter. The host actin was labeled specifically by the red fluorescence substance, tetramethy rhodamine isothicyanete-phalloidin. Observation under a fluorescence microscopy showed that VP39, which was indicated by green fluorescence, began to appear in the cells 6 h after being transfected with pEGFP39. Red actin cables were also formed in the cytoplasm at the same time. Actin was aggregated in the nucleus 9 h after the transfection. The green and red fluorescence always appeared in the same location of the cells, which demonstrated that VP39 could combine with the host actin. Such a combination would result in the actin skeleton rearrangement.

• ALGAE
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• v.37 no.1
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• pp.75-84
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• 2022

Intercellular nutrient and signal transduction are essential to sustaining multicellular organisms and maximizing the benefits of multicellularity. It has long been believed that red algal intercellular transport of macromolecules is prevented by the protein-rich pit plug within pit-connections, the only physical connection between cells. Fluorescein isothiocyanate-dextran and recombinant green fluorescence protein (rGFP) of various molecular sizes were injected into vegetative cells of Griffithsia monilis using a micromanipulator, and intercellular transport of the fluorescent probes was examined. Pit-connections were found to provide intercellular transport of tracers at rates comparable to plasmodesmata in other organisms. The time necessary for the transport to an adjacent cell was dependent on the molecular size and the direction of the transport. Fluorescent dextran of 3 kDa was transported to adjacent cells in 1-2 h after injection and migrated to all cells of the filament within 24 h, but fluorescent dextran of 10-20 kDa took 24 h to transfer to neighboring cells. The migration occurred faster towards adjacent reproductive cells and to apical cells than basally. Fluorescent tracers above 40 kDa and rGFP was not transported to neighboring cells, but accumulated near the pit plug. Our results suggest that pit-connections are conduit for macromolecules between neighboring cells and that these size-specific conduits allow intercellular communication between the vegetative cells of red algae.

• Journal of Sericultural and Entomological Science
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• v.50 no.2
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• pp.87-92
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• 2012

We constructed the fibroin H-chain expression system to produce Discosoma sp. red fluorescent protein variant2 (DsRed2) in transgenic silkworm cocoon. Fluorescent cocoon could be made by fusing DsRed2 cDNA to the heavy chain gene and injecting it into a silkworm. The DsRed2 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 DsRed2/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworms. The EGFP fluorescence became visible in the ocelli and in the central and peripheral nervous system on the seventh day of embryonic development. A mixture of the donor and helper vector was micro-injected into 1,020 Kumokjam, bivoltin silkworm eggs. We obtained 6 broods. The cocoon was displayed strong red fluorescence, proving that the fusion protein was present in the cocoon. Accordingly, we suggest that the DsRed2 fluorescence silk will enable the production of novel biomaterial based on the transgenic silk.

• Journal of Microbiology and Biotechnology
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• v.25 no.4
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• pp.503-510
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• 2015

The iLOV protein belongs to a family of blue-light photoreceptor proteins containing a light-oxygen-voltage sensing domain with a noncovalently bound flavin mononucleotide (FMN) as its chromophore. Owing to advantages such as its small size, oxygen-independent nature, and pH stability, iLOV is an ideal candidate over other reporter fluorescent proteins such as GFP and DsRed. Here, for the first time, we describe the feasibility of applying LOV domain-based fluorescent iLOV as a metal sensor by measuring the fluorescence quenching of a protein with respect to the concentration of metal ions. In the present study, we demonstrated the inherent copper sensing property of the iLOV protein and identified the possible amino acids responsible for metal binding. The fluorescence quenching upon exposure to Cu²⁺ was highly sensitive and exhibited reversibility upon the addition of the metal chelator EDTA. The copper binding constant was found to be 4.72 ± 0.84 µM. In addition, Cu²⁺-bound iLOV showed high fluorescence quenching at near physiological pH. Further computational analysis yielded a better insight into understanding the possible amino acids responsible for Cu²⁺ binding with the iLOV protein.

• 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.26 no.3
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• pp.530-539
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• 2016

Bacterial light-oxygen-voltage-sensing photoreceptor-derived flavin mononucleotide (FMN)-based fluorescent proteins act as a promising distinct class of fluorescent proteins utilized for various biomedical and biotechnological applications. The key property of its independency towards oxygen for its chromophore maturation has greatly helped this protein to outperform the other fluorescent proteins such as GFP and DsRed for anaerobic applications. Here, we describe the feasibility of FMN-containing fluorescent protein FbFP as a metal-sensing probe by measuring the fluorescence emission changes of a protein with respect to the concentration of metal ions. In the present study, we demonstrated the mercury-sensing ability of FbFP protein and the possible amino acids responsible for metal binding. A ratiometric approach was employed here in order to exploit the fluorescence changes observed at two different emission maxima with respect to Hg²⁺ at micromolar concentration. The engineered variant FbFP_C56I showed high sensitivity towards Hg²⁺ and followed a good linear relationship from 0.1 to 3 μM of Hg²⁺. Thus, further engineering with a rational approach would enable the FbFP to be developed as a novel and highly selective and sensitive biosensor for other toxic heavy metal ions as well.

• KSBB Journal
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• v.30 no.6
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• pp.296-301
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• 2015

Eggshell-based biocomposites have become attractive due to their exquisite nanostructure and biological properties, which are mainly composed of highly organized calcium carbonate crystals controlled by organic macromolecules such as proteins and polysaccharides. Here, we designed the recombinant fusion protein of a putative eggshell matrix protein named as GG1234 and a fluorescent reporter protein of DsRed. The protein was successfully over-expressed in E. coli and purified by Ni-NTA affinity chromatography. In vitro calcium carbonate crystallization was conducted in the presence of the fusion protein, and morphological change was investigated. The protein inhibited the calcite growth in vitro, and spherical calcium carbonate micro-particles with the diameter of about $20-30{\mu}m$ were obtained. We expect that this study would be helpful for better understanding of eggshell-based biomineralization.

• Journal of Ginseng Research
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• v.43 no.1
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• pp.86-94
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• 2019

Background: Ginseng is believed to have antitumor activity. Autophagy is largely a prosurvival cellular process that is activated in response to cellular stressors, including cytotoxic chemotherapy; therefore, agents that inhibit autophagy can be used as chemosensitizers in cancer treatment. We examined the ability of Korean Red Ginseng extract (RGE) to prevent autophagic flux and to make hepatocellular carcinoma (HCC) cells become more sensitive to doxorubicin. Methods: The cytotoxic effects of total RGE or its saponin fraction (RGS) on HCC cells were examined by the lactate dehydrogenase assay in a dose- or time-dependent manner. The effect of RGE or RGS on autophagy was measured by analyzing microtubule-associated protein 1A/1B-light chain (LC)3-II expression and LC3 puncta formation in HCC cells. Late-stage autophagy suppression was tested using tandem-labeled green fluorescent protein (GFP)-monomeric red fluorescent protein (mRFP)-LC3. Results: RGE markedly increased the amount of LC3-II, but green and red puncta in tandem-labeled GFP-mRFP-LC3 remained colocalized over time, indicating that RGE inhibited autophagy at a late stage. Suppression of autophagy through knockdown of key ATG genes increased doxorubicin-induced cell death, suggesting that autophagy induced by doxorubicin has a protective function in HCC. Finally, RGE and RGS markedly sensitized HCC cells, (but not normal liver cells), to doxorubicin-induced cell death. Conclusion: Our data suggest that inhibition of late-stage autophagic flux by RGE is important for its potentiation of doxorubicin-induced cancer cell death. Therapy combining RGE with doxorubicin could serve as an effective strategy in the treatment of HCC.

• Journal of Sericultural and Entomological Science
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• v.51 no.2
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• pp.153-158
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• 2013

To express green fluorescent protein in the cocoon of silkworm, we constructed the fibroin H-chain expression system to produce enhanced green fluorescent protein (EGFP) in the cocoon of transgenic silkworms. The EGFP 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 EGFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3-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 1,200 eggs of bivoltin silkworms, Baegokjam. We obtained 8 broods. The cocoon displayed strong green 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 immunoblotting. Accordingly, we suggest that the EGFP fluorescence silk will enable the production of the novel biomaterial based on the transgenic silk.