• Molecules and Cells
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• v.21 no.2
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• pp.244-250
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• 2006

Gangliosides are receptors for various peptides and proteins including neuropeptides, ${\beta}$-amyloid proteins, and prions. Recently, the role of gangliosides in mucosal immunization has attracted attention due to the emerging interest in oral vaccination. Ganglioside GM1 exists in abundance on the surface of the M cells of Peyer's patch, a well-known mucosal immunity induction site. In the present study we identified a peptide ligand for GM1 and tested whether it played a role in immune induction. GM1-binding peptides were selected from a phage-displayed dodecapeptide library and one peptide motif, GWKERLSSWNRF, was fused to the C-terminus of enhanced green fluorescent protein (EGFP). The fusion protein, but not EGFP fused with a control peptide, was concentrated around Peyer's patch after incubation in the lumen of the intestine ex vivo. Furthermore, oral feeding of the fusion protein but not control EGFP induced mucosal and systemic immune responses against EGFP resembling Th2-type immune responses.

• Proceedings of the Korean Society of Sericultural Science Conference
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• 2003.04a
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• pp.40-40
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• 2003

Recombinant plasmids harboring heterologous genes coding enhanced green fluorescent protein (EGFP) and erythropoietin (EPO) were transfected and expressed in Drosophila melanogaster S2 cells. Stably transformed cell populations expressing EGFP or monkey EPO were isolated after 4 weeks of selection with hygromycin B. (omitted)

• International Journal of Industrial Entomology and Biomaterials
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• v.15 no.1
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• pp.35-38
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• 2007

In this study, ORF 46 of Bombyx mod nucleopolyhedrovirus(Bm46) fused with EGFP was expressed in Bombyx mod cell lines, larvae and pupae by BmNPV Bacmid system. Bm46 and EGFP were cloned into donor plasmid pFastBacHTb, which was transformed to competent DH10B cells containing helper and BmNPV bacmid by site-specific transposition. Recombinant bacmid was used to transfected BmN-4 cells to produce the recombinant baculovirus vBm-Bm46-EGFP. Recombination virus was injected into silkworm larvae and pupae. The expression of the fusion protein was monitored by examining green fluorescence using a fluorescent microscope. Intense fluorescence in cells and silkworm was observed at 4 days post-infection, indicating the Bm46-EGFP fusion gene was expressed successfully.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.91-91
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• 2002

There are many methods to introduce exogenous DNA into embryo for the purpose of producing transgenic animals. Exogenous gene can be integrated into oocyte as a form of sperm vector. In this study, sperm was used as a vector for transgene that is enhanced green fluorescent protein (EGFP). The objective of this study was to investigate the expression of exogenous gene in bovine embryos after injection of spermatozoa cocultured with EGFP fragment. Spermatozoa were plunged into liquid nitrogen and thawed several times or shaked in 0.2% Triton X-100 to remove sperm membrane which followed by DTT treatment. (omitted)

• BMB Reports
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• v.40 no.5
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• pp.611-616
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• 2007

$E^{rns}$ is an envelope glycoprotein of classical swine fever virus (CSFV) and has an unusual feature of RNase activity. In the present study, we demonstrate that $E^{rns}$ counteracts Newcastle disease virus (NDV)-mediated induction of IFN-$\beta$. For this purpose, $E^{rns}$ fused to the enhanced green fluorescent protein (EGFP) was transiently expressed in porcine kidney 15 (PK15) cells. In luciferase activity assay, $E^{rns}$-EGFP was found to prevent IFN-$\beta$ promoter-driven luciferase expression and block the induction of IFN-$\beta$ promoter mediated by NDV in a dose-dependent manner. Through IFN-specific semi-quantitative RT-PCR detection, obvious decrease of IFN-$\beta$ mRNA in NDV-infected PK15 cells was observed in the presence of $E^{rns}$-EGFP. In contrast, EGFP alone showed none of this block capacity. In addition, $E^{rns}$-EGFP mutations with RNase inactivation were also found to block NDV-mediated induction of IFN-$\beta$. These evidences establish a novel function for CSFV $E^{rns}$ glycoprotein in counteraction of the IFN-$\beta$ induction pathway.

• Journal of Microbiology and Biotechnology
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• v.24 no.2
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• pp.144-151
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• 2014

Increasing the gene copy number has been commonly used to enhance the protein expression level in the yeast Pichia pastoris. However, this method has been shown to be effective up to a certain gene copy number, and a further increase of gene dosage can result in a decrease of expression level. Evidences indicate the gene dosage effect is product-dependent, which needs to be determined when expressing a new protein. Here, we describe a direct detection of the gene dosage effect on protein secretion through expressing the enhanced green fluorescent protein (EGFP) gene under the direction of the ${\alpha}$-factor preprosequence in a panel of yeast clones carrying increasing copies of the EGFP gene (from one to six copies). Directly examined under fluorescence microscopy, we found relatively lower levels of EGFP were secreted into the culture medium at one copy and two copies, substantial improvement of secretion appeared at three copies, plateau happened at four and five copies, and an apparent decrease of secretion happened at six copies. The secretion of EGFP being limiting at four and five copies was due to abundant intracellular accumulation of proteins, observed from the fluorescence image of yeast and confirmed by western blotting, which significantly activated the unfolded protein response indicated by the up-regulation of the BiP (the KAR2 gene product) and the protein disulfide isomerase. This study implies that tagging a reporter like GFP to a specific protein would facilitate a direct and rapid determination of the optimal gene copy number for high-yield expression.

• BMB Reports
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• v.45 no.4
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• pp.227-232
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• 2012

In vertebrates, there are two variants of eukaryotic peptide elongation factor 1A (eEF1A; formerly eEF-$1{\alpha}$), eEF1A1 and eEF1A2, which have three well-conserved domains ($D_I$, $D_{II}$, and $D_{III}$). In neurons, eEF1A1 is the embryonic type, which is expressed during embryonic development as well as the first two postnatal weeks. In the present study, EGFP-tagged eEF1A1 truncates were expressed in cortical neurons isolated from rat embryo (E18-19). Live cell images of transfected neurons showed that $D_{III}$-containing EGFP-fusion proteins (EGFP-$D_{III}$, -$D_{II-III}$, -$D_{I-III}$) formed clusters that were confined within somatodendritic domains, while $D_{III}$-missing ones (EGFP-$D_I$, -$D_{II}$, -$D_{I-II}$) and control EGFP were homogeneously dispersed throughout the neuron including axons. In dendrites, EGFP-$D_{III}$ was targeted to the heads of spine- and filopodia-like protrusions, where it was colocalized with $SynGAP{\alpha}$, a postsynaptic marker. Our data indicate that $D_{III}$ of eEF1A1 mediates formation of clusters and localization to spines.

• Parasites, Hosts and Diseases
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• v.53 no.4
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• pp.385-394
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• 2015

Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-${\gamma}$/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency.

• Journal of Life Science
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• v.28 no.2
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• pp.162-169
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• 2018

Many cytoplasmic proteins are targeted to the cytoplasmic membrane of the trans-Golgi network (TGN) via an N-terminal short helix. We previously showed that the 20 N-terminal amino acids of Aplysia phosphodiesterase 4 (ApPDE4) long form are sufficient for its targeting to the plasma membrane and the TGN. The N-terminus of the ApPDE4 long form binds to PI4P and sulfatide in vitro. Therefore, in order to decipher the roles of sulfatide in Golgi complex targeting, we examined the cellular localization of sulfatide-binding peptides. In this study, we found that enhanced green fluorescent protein (EGFP) fused to the C-terminus of modified sulfatide- and heparin-binding peptides (mHSBP-EGFP) was localized to the TGN. On the other hand, its mutant, in which tryptophan was replaced with an alanine, leading to the impairment of heparin and sulfatide binding, was localized to cytosol. We also found that the TGN targeting of mHSBP-EGFP is impaired by the treatment of antimycin A, phenylarsine oxide (PAO), and adenosine but not a high concentration of wortmannin. These results suggest that PAO and adenosine-sensitive kinases, including phosphatidylinositol 4-kinase II, may play key roles in the recruitment of mHSBP-EGFP.

• 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.