• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1022-1027
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• 2005

Based on plasmid display technology by the complexes of fusion protein and the encoding plasmid DNA, an in vitro selection method for high affinity DNA-binding protein was developed and experimentally demonstrated. The GAL4 DNA-binding domain (GAL4 DBD) was selected as a model DNA-binding protein, and enhanced green fluorescent protein (EGFP) was used as an expression reporter for the selection of target proteins. Error prone PCR was conducted to construct a mutant library of the model. Based on the affinity decrease with increased salt concentration, mutants of GAL4 DBD having high affinity were selected from the mutant protein library of protein-encoding plasmid complex by this method. Two mutants of (Lys33Glu, Arg123Lys, Ile127Lys) and (Ser47Pro, Ser85Pro) having high affinity were obtained from the first generation mutants. This method can be used for rapid in vitro selection of high affinity DNA-binding proteins, and has high potential for the screening of high affinity DNA-binding proteins in a sequence-specific manner.

• 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.

• 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 Microbiology and Biotechnology
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• v.24 no.2
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• pp.152-159
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• 2014

The regulation of protein tyrosine phosphorylation is mediated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) and is essential for cellular homeostasis. Co-expression of PTKs with PTPs in Pichia pastoris was used to facilitate the expression of active PTKs by neutralizing their apparent toxicity to cells. In this study, the gene encoding phosphatase PTP1B with or without a blue fluorescent protein or peroxisomal targeting signal 1 was cloned into the expression vector pAG32 to produce four vectors. These vectors were subsequently transformed into P. pastoris GS115. The tyrosine kinases EGFR-2 and $PDGFR{\beta}$ were expressed from vector pPIC3.5K and were fused with a His-tag and green fluorescent protein at the N-terminus. The two plasmids were transformed into P. pastoris with or without PTP1B, resulting in 10 strains. The EGFR-2 and $PDGFR{\beta}$ fusion proteins were purified by $Ni^{2+}$ affinity chromatography. In the recombinant P. pastoris, the PTKs co-expressed with PTP1B exhibited higher kinase catalytic activity than did those expressing the PTKs alone. The highest activities were achieved by targeting the PTKs and PTP1B into peroxisomes. Therefore, the EGFR-2 and $PDGFR{\beta}$ fusion proteins expressed in P. pastoris may be attractive drug screening targets for anticancer therapeutics.

• Journal of Microbiology and Biotechnology
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• v.22 no.9
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• pp.1271-1278
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• 2012

Genetic fusion of two proteins frequently induces beneficial effects to the proteins, such as increased solubility, besides the combination of two protein functions. Here, we study the effects of the bacterial surface layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a on the folding of a C-terminally fused enhanced green fluorescent protein (EGFP) moiety. Although GFPs are generally unable to adopt a functional confirmation in the bacterial periplasm of Escherichia coli cells, we observed periplasmic fluorescence from a chimera of a 150-amino-acid N-terminal truncation of SgsE and EGFP. Based on this finding, unfolding and refolding kinetics of different S-layer-EGFP chimeras, a maltose binding protein-EGFP chimera, and sole EGFP were monitored using green fluorescence as indicator for the folded protein state. Calculated apparent rate constants for unfolding and refolding indicated different folding pathways for EGFP depending on the fusion partner used, and a clearly stabilizing effect was observed for the SgsE_C fusion moiety. Thermal stability, as determined by differential scanning calorimetry, and unfolding equilibria were found to be independent of the fused partner. We conclude that the stabilizing effect SgsE_C exerts on EGFP is due to a reduction of degrees of freedom for folding of EGFP in the fused state.

• Molecules and Cells
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• v.23 no.3
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• pp.357-362
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• 2007

There is an increasing demand for high throughput (HTP) methods for gene analysis on a genome-wide scale. However, the current repertoire of HTP detection methodologies allows only a limited range of cellular phenotypes to be studied. We have constructed two HTP-optimized expression vectors generated from the red fluorescent reporter protein (RFP) gene. These vectors produce RFP-tagged target proteins in a multiple expression system using gateway cloning technology (GCT). The RFP tag was fused with the cloned genes, thereby allowing us localize the expressed proteins in mammalian cells. The effectiveness of the vectors was evaluated using an HTP-screening system. Sixty representative human C2 domains were tagged with RFP and overexpressed in HiB5 neuronal progenitor cells, and we studied in detail two C2 domains that promoted the neuronal differentiation of HiB5 cells. Our results show that the two vectors developed in this study are useful for functional gene analysis using an HTP-screening system on a genome-wide scale.

• Applied Chemistry for Engineering
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• v.32 no.1
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• pp.10-14
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• 2021

mCherry is one of the well-understood red fluorescent proteins which has a similar tertiary structure as GFPs, but pH resistant due to the lack of hydrogen bond network. Whereas mCherry-I202T showed far-red fluorescence and also pH sensitive property because of the additional hydrogen bond formed by substituting Ile of 202 amino acid sequence on mCherry with Thr. In order to verify the pH sensitive characteristic of mCherry-I202T owing to the extension of hydrogen bond, UV-vis spectrum was measured over the range of acidic to basic pH. We also demonstrate further possibilities of applying mCherry-I202T as a pH sensor.

• The Korea Journal of Herbology
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• v.30 no.2
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• pp.25-30
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• 2015

Objectives : Laver (Porphyra tenera), a red algae species, is one of the most widely consumed edible seaweed in Korea. Laver contains various substances such as essential amino acid, fiber, minerals and polyphenols that benefit human health. In the present study, we prepared ethanol extracts from commercially processed product of Porphyra tenera, and evaluated the growth inhibitory effect against human oral squamous carcinoma YD-10B cells. Methods : Cell viability was measured by MTT assay. Apoptosis was confirmed by TUNEL assay and flow cytometry with the green fluorescent dye FITC annexin V entering apoptotic cells and the red fluorescent dye PI not entering. The expression of the relevant proteins was detected using Western blot. Results : Ethanol extracts of Porphyra tenera (PTE, $50-200{\mu}g/m{\ell}$) caused a significant decrease of cell viability in a dose dependant manner. The cell death occurred as a result of apoptotic process as determined by TUNEL assay and flow cytometric analysis. In line with this observation, decrease in procaspase proteins and increase in cytosolic cytochrome c were observed in cells treated with PTE. In addition, exposure to PTE decreased the expression levels of Bcl-2, and induced PARP cleavage and AIF translocation from mitochondria to nucleus. Conclusions : In conclusion, PTE exerts anti-cancer effects by inducing apoptosis via caspase activation and AIF nuclear translocation in YD-10B cells. These results provide evidence for the possible therapeutic effect of Porphyra tenera in oral cancer cells.

• KSBB Journal
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• v.29 no.5
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• pp.328-335
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• 2014

Vehicle toxicity is one of the main obstacles for intracellular delivery of bioactive compounds. Silk fibroin is a natural polymer proven to have high biocompatibility since being used as suture material. In this report, fibroin microspheres were prepared without any chemical modification or cross-linking not to affect its biocompatibility. The microspheres were taken up by more than 90% of 3T3 cells. Cellular uptake continued after medium replenishment with a different-colored fluorescent microsphere, suggesting that simultaneous ingestion and exocytosis occurred. Cellular uptake of fibroin microspheres did not affect cell viability. Intracellular trafficking of the microspheres using lysosome-specific fluorescent dye revealed that fibroin microspheres were localized both in the cytoplasm and in the lysosome. Accordingly, fibroin microspheres can be a potential vehicle for intracytoplasmic delivery of large cargos, such as mixtures of proteins, nutrients or artificial organelles.

• Journal of Microbiology and Biotechnology
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• v.14 no.6
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• pp.1333-1337
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• 2004

Oxidants such as hydrogen peroxide are powerful inducers of cell damage, ageing, and apoptosis. Since clusterin, a 75-80 kDa mammalian glycoprotein, is frequently found to be inducible in apoptotic cells and tissues, this study inquired into whether this would be a protective mechanism against further cell death. The aim was to find out whether overexpression of clusterin could protect cells from oxidant­induced stress and apoptosis. To clarify this issue, we generated and analyzed stable cell lines expressing fusion proteins of a rat clusterin with an enhanced green fluorescent protein (EGFP). When treated with varying concentrations of hydrogen peroxides, clusterin transfectants indeed showed increased resistance to apoptosis and exhibited a much higher survival rate than mock-transfected cells. On the other hand, neither intracellular re-distribution nor local concentration of clusterin-EGFP was observed, which leaves the question open about its anti-apoptotic mechanism. In conclusion, the overexpression of clusterin provides a means for protecting cells against oxidative stress and subsequent cell death.