• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.3
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• pp.275-279
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• 2005

Insect cell transformants, stably expressing human $GFP_{uv}-{\beta}1$, 3-N-acetylglucosaminyltransferase 2 $({\beta}3GnT2)$ as the green fluorescent protein $(GFP_{uv})-fused$ protein, were efficiently isolated on Western blot by the quantification of the densitometric intensity of the fusion protein. From almost 150 transformants containing the fusion gene linked to three different types of signal sequence, two transformants, Tn-pXme4a and -pX28a, were successfully selected, showing 8.3 and 8.6 mU/mL ${\beta}3GnT$ activity, respectively. This method requires a screening time almost one-half that required in the isolation of stably transformed cells with high expression levels, and at the same time allows the handling a large number of transformants.

• Clinical and Experimental Reproductive Medicine
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• v.40 no.1
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• pp.1-6
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• 2013

Objective: Oocyte-specific homeobox 4 (Obox4) is preferentially expressed in oocytes and plays an important role in the completion of meiosis of oocytes. However, the Obox4 expression pattern has not been reported yet. In this study, we investigated the subcellular localization of Obox4 using a green fluorescent protein (GFP) fusion expression system. Methods: Three regions of Obox4 were divided and fused to the GFP expression vector. The partly deleted homeodomain (HD) regions of Obox4 were also fused to the GFP expression vector. The recombinant vectors were transfected into HEK-293T cells plated onto coated glass coverslips. The transfected cells were stained with 4',6-diamidino-2-phenylindol and photographed using a fluorescence microscope. Results: Mutants containing the HD region as well as full-length Obox4 were clearly localized to the nucleus. In contrast, the other mutants of either the N-terminal or C-terminal region without HD had impaired nuclear localization. We also found that the N-terminal and C-terminal of the Obox HD contributed to nuclear localization and the entire HD was necessary for nuclear localization of Obox4. Conclusion: Based on the results of the present study, we demonstrated that the intact HD region of Obox4 is responsible for the nuclear localization of Obox4 protein in cells.

• Parasites, Hosts and Diseases
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• v.45 no.3
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• pp.165-174
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• 2007

Toxoplasma gondii GRA10 expressed as a GFP-GRA10 fusion protein in HeLa cells moved to the nucleoli within the nucleus rapidly and entirely. GRA10 was concentrated specifically in the dense fibrillar component of the nucleolus morphologically by the overlap of GFP-GRA10 transfection image with IFA images by monoclonal antibodies against GRA10 (Tg378), B23 (nucleophosmin) and C23 (nucleolin). The nucleolar translocalization of GRA10 was caused by a putative nucleolar localizing sequence (NoLS) of GRA10. Interaction of GRA10 with TATA-binding protein associated factor 1B (TAF1B) in the yeast two-hybrid technique was confirmed by GST pull-down assay and immunoprecipitation assay. GRA10 and TAF1B were also co-localized in the nucleolus after co-transfection. The nucleolar condensation of GRA10 was affected by actinomycin D. Expressed GFP-GRA10 was evenly distributed over the nucleoplasm and the nucleolar locations remained as hollows in the nucleoplasm under a low dose of actinomycin D. Nucleolar localizing and interacting of GRA10 with TAF1B suggested the participation of GRA10 in rRNA synthesis of host cells to favor the parasitism of T. gondii.

• BMB Reports
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• v.42 no.5
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• pp.281-285
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• 2009

Peroxisomes play an important role in cellular defense systems and generate secondary messengers for cellular communication. Saccharomyces cerevisiae containing oleate-induced peroxisomes were subjected to buffer-soluble extraction and two chromatographic procedures, and a protein with antifungal activity was isolated. The results of MALDI-TOF analysis identified the isolated protein as peroxisomal 3-ketoacyl-CoA thiolase (ScFox3). Purified yeast ScFox3 exhibited thiolase activity that catalyzed the thiolytic cleavage of 3-ketoacyl-CoA to acetyl-CoA and acyl-CoA. ScFox3 protein inhibited various pathogenic fungal strains, with the exception of Aspergillus flavus. Using ScFox3-GFP and PTS2 signal-truncated ScFox3M-GFP, we showed that only ScFox3-GFP, with an intact PTS2 peroxisome signal sequence, was able to translocate into peroxisomes. Yeast ScFox3 is a natural antifungal agent found in peroxisomes.

• Journal of Microbiology and Biotechnology
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• v.15 no.1
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• pp.141-146
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• 2005

A new system for displaying proteins on the surface of Escherichia coli was developed using the Salmonella typhimurium outer membrane protein C (OmpC) as an anchoring motif. The C-terminal deletionfusion strategy was developed to fuse the polyhistidine peptides and green fluorescent protein (GFP) to the Cterminal of the truncated functional portion of OmpC. The polyhistidine peptides of up to 243 amino acids could besuccessfully displayed on the E. coli cell surface, which allowed recombinant E. coli to adsorb up to 34.2 μmol of Cd2+ per gram dry cell weight. The GFP could also be successfully displayed on the E. coli cell surface. These results suggest that the C-terminal deletion-fusion strategy employing the S. typhimurium OmpC as an anchoring motif provides a new efficient way for the display of large proteins on the surface of E. coli.

• The Plant Pathology Journal
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• v.32 no.4
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• pp.371-376
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• 2016

Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH) gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana.

• Molecules and Cells
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• v.26 no.3
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• pp.270-277
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• 2008

This study addresses the physiological functions of the Ran-binding protein homolog NbRanBP1 in Nicotiana benthamiana. Virus-induced gene silencing (VIGS) of NbRanBP1 caused stunted growth, leaf yellowing, and abnormal leaf morphology. The NbRanBP1 gene was constitutively expressed in diverse tissues and an NbRanBP1:GFP fusion protein was primarily localized to the nuclear rim and the cytosol. BiFC analysis revealed in vivo interaction between NbRanBP1 and NbRan1 in the nuclear envelope and the cytosol. Depletion of NbRanBP1 or NbRan1 reduced nuclear accumulation of a NbBTF3:GFP marker protein. In the later stages of development, NbRanBP1 VIGS plants showed stress responses such as reduced mitochondrial membrane potential, excessive production of reactive oxygen species, and induction of defense-related genes. The molecular role of RanBP1 in plants is discussed in comparison with RanBP1 function in yeast and mammals.

• YAKHAK HOEJI
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• v.57 no.4
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• pp.265-271
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• 2013

Treatments of vascular disease via modulating the expression of specific proteins by gene transfer have been attempted in various studies over the past few years. Among several methods to deliver genes, adenovirus currently has been used because of a number of positive aspects. In this study, we test adenoviral vector as a potential mediator in the treatment of vascular disease by using freshly isolated vascular tissues not cultured vascular cells. Freshly isolated vascular tissues were directly exposed to adenoviral vector pAd5CMVmcsIRESeGFPpA to check the possibility of GFP expression in different layer of vascular tissues. We found that the GFP expression by using adenoviral vector experiments is mainly focused on the adventitia and failed to detect GFP expression at endothelial layer or vascular smooth muscle layer in vascular tissues. However, we also found that several integrin receptors are robustly expressed in vascular smooth muscle, thus the limited expression of protein in vascular smooth muscle are not likely the lack of integrin receptors. In conclusion, adenovirus could not be a good tool for a specific protein expression in vascular smooth muscle cell. Thus, the application of adenovirus as a tool for gene therapy of vascular smooth muscle cells in clinical therapeutic trial need to be optimized further.

• 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.

• The Plant Pathology Journal
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• v.35 no.1
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• pp.19-31
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• 2019

Bud rot (BR) is the most devastating disease affecting oil palm (Elaeis guineensis) crops in Colombia. Its causal agent, Phytophthora palmivora, initiates the infection in immature oil palm leaflets producing necrotic lesions, followed by colonization of opportunistic necrotrophs, which increases disease damage. To improve the characterization of the disease, we transformed P. palmivora using Agrobacterium tumefaciens-mediated transformation (ATMT) to include the fluorescent proteins CFP-SKL (peroxisomal localization), eGFP and mRFP1 (cytoplasmic localization). The stability of some transformants was confirmed by Southern blot analysis and single zoospore cultures; additionally, virulence and in vitro growth were compared to the wild-type isolate to select transformants with the greatest resemblance to the WT isolate. GFP-tagged P. palmivora was useful to identify all of the infective structures that are commonly formed by hemibiotrophic oomycetes, including apoplastic colonization and haustorium formation. Finally, we detected cell death responses associated with immature oil palm tissues that showed reduced susceptibility to P. palmivora infection, indicating that these tissues could exhibit age-related resistance. The aim of this research is to improve the characterization of the initial disease stages and generate cell biology tools that may be useful for developing methodologies for early identification of oil palm materials resistant or susceptible to BR.