• Journal of Plant Biotechnology
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• v.43 no.4
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• pp.432-437
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• 2016

Interleukin-11 (IL-11) is a cytokine that plays a key regulatory role in the immune system. Recombinant human IL-11 (rhIL-11) exerts a preventative effect against apoptotic cell death and inhibits preadipocyte differentiation. IL-11 also is used to stimulate the bone marrow to produce platelets in order to prevent low platelets that may be caused by chemotherapy. Unfortunately, the high production cost of IL-11 associated. In this study, we investigated the feasibility of transgenic plants for the cost-effective production of rhIL-11. Production of rhIL-11 proteins in whole-plant expression system will be more economical when compared to the current E. coli based expression system. The human rhIL-11 gene was codon optimized to maximize plant host system expression. IL-11 expression vector under the control of a constitutive cauliflower mosaic virus 35S (CaMV 35S) promoter was introduced into tobacco by Agrobacterium-mediated transformation. The 5'-leader sequence (called ${\Omega}$) of tobacco mosaic virus (TMV) as a translational enhancer was added to construct. Transgenic tobacco plants expressing various levels of rhIL-11 protein were generated. Western blotting of the stably transformed lines demonstrated accumulation of the appropriately sized rhIL-11 protein in leaves. This research demonstrated the efficacy of using tobacco as an expression system for the production of rhIL-11.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.85-85
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• 2003

Human lactoferrin (hLF) is an 80 kDa iron-binding glycoprotein that is expressed in high concentration in milk and in lesser amount in the secondary or specific granules of neutrophils and in plasma, LF is classically considered to be related to the binding, transport, and storage of iron. The transgenic mice carrying the human hLF gene in conjunction with the bovine $\beta$-casein promoter produced the human hLF in their milk during lactation. To screen transgenic mice, PCR was carried out using chromosomal DNA extracted from tail or toe tissues. In this study, stability of germ line transmission and expression of hLF were monitored up to generation Fl7 of a transgenic line. When female mouse of generation F9 was crossbred with normal male, generation F9 to Fl7 mice showed similar transmission rates ($66.0 \pm 12.57%, 42.0 \pm 14.98%, 72.2 \pm 25.45%, 50.0 \pm 16.70%, 65.7 \pm 6.45%, 48.6 \pm 14.65%, 54 1 \pm 18 11%, 57.8 \pm 16.16% and 48.6 \pm 20.66$, respectively), implying that the hLF gene can be transmitted stably up to long term generation in the transgenic mice For ELISA analysis, hLF expression levels were determined with an hLF ELISA kit in accordance with the supplier's protocol. Expression levels of human hLF from milk of generation F9 to Fl3 mice were $ 3.2 \pm 0.69 mg/ml, 3.1 \pm 0.81 mg/ml, 4.6 \pm 1.38 mg/ml, 3.1 \pm 0.42 mg/ml, and 4.5 \pm 1,48 mg/ml$, respectively. These expression levels were lower than that of founder (6.6 mg/$m\ell$) mouse. We concluded that transgenic mice faithfully passed the transgene on their progeny and successively secreted target proteins into their milk through several generations.

• Journal of Microbiology and Biotechnology
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• v.24 no.2
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• pp.197-208
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• 2014

A novel protease gene from Bacillus gibsonii, aprBG, was cloned, expressed in B. subtilis, and characterized. High-level expression of aprBG was achieved in the recombinant strain when a junction was present between the promoter and the target gene. The purified recombinant enzyme exhibited similar N-terminal sequences and catalytic properties to the native enzyme, including high affinity and hydrolytic efficiency toward various substrates and a superior performance when exposed to various metal ions, surfactants, oxidants, and commercial detergents. AprBG was remarkably stable in 50% organic solvents and retained 100% activity and stability in 0-4 M NaCl, which is better than the characteristics of previously reported proteases. AprBG was most closely related to the high-alkaline proteases of the subtilisin family with a 57-68% identity. The secretion and maturation mechanism of AprBG was dependent on the enzyme activity, as analyzed by site-directed mutagenesis. Thus, when taken together, the results revealed that the halo-solvent-tolerant protease AprBG displays significant activity and stability under various extreme conditions, indicating its potential for use in many biotechnology applications.

• Journal of Plant Biotechnology
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• v.32 no.3
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• pp.209-215
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• 2005

Lactoferrin is an iron-binding glycoprotein with many biological roles, including the protection against microbial and virus infection, stimulation of the immune system. We developed the transgenic Siberian ginseng (Acanthopanax senticosus) cell cultures producing the human lactoferrin (hLf) protein following Agrobacterium tumefaciens-mediated transformation. A construct containing a targeting signal peptide from tobacco endoplasmic reticulum fused to hLf cDNA under the control of an oxidative stress-inducible SWPA2 promoter was engineered. Transgenic Siberian ginseng cultured cells to produce a recombinant hLf protein were successfully generated and confirmed by PCR and Southern blot analysis. ELISA and western blot analysis showed that full length-hLf protein was synthesized in the transgenic cells. The production of hLf increased proportionally to cell growth and reached a maximal (up to 3% of total soluble proteins) at the stationary phase. These results suggest that the transgenic Siberian ginseng cultured cells in this study will be biotechnologically useful for the commercial production of medicinal plant cell cultures to produce hLf protein.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1563-1570
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• 2010

To construct a highly sensitive detection system for endocrine disruptors (EDs), we have compared the activity of promoters with the n-alkane-inducible cytochrome P450 gene (ALK1), isocitrate lyase gene (ICL1), ribosomal protein S7 gene (RPS7), and the translation elongation factor-1${\alpha}$ gene (TEF1) for the heterologous gene in Yarrowia lipolytica. The promoters were introduced into the upstream of the lacZ or hERa reporter genes, respectively, and the activity was evaluated by ${\beta}$-galactosidase assay for lacZ and Western blot analysis for hER${\alpha}$. The expression analysis revealed that the ALK1 and ICL1 promoters were induced by n-decane and by EtOH, respectively. The constitutive promoter of RPS7 and TEF1 showed mostly a high level of expression in the presence of glucose and glycerol, respectively. In particular, the TEF1 promoter showed the highest ${\beta}$-galactosidase activity and a significant signal by Western blotting with the anti-estrogen receptor, compared with the other promoters. Moreover, the detection system was constructed with promoters linked to the upstream of the expression vector for the hER${\alpha}$ gene transformed into the Y. lipolytica with a chromosome-integrated lacZ reporter gene under the control of estrogen response elements (EREs). It was indicated that a combination of pTEF1p-hER${\alpha}$ and CXAU1-2XERE was the most effective system for the $E_2$-dependent induction of the ${\beta}$-galactosidase activity. This system showed the highest ${\beta}$-galactosidase activity at $10^{-6}\;M\;E_2$, and the activity could be detected at even the concentration of $10^{-10}\;M\;E_2$. As a result, we have constructed a strongly sensitive detection system with Y. lipolitica to evaluate recognized/suspected ED chemicals, such as natural/synthetic hormones, pesticides, and commercial chemicals. The results demonstrate the utility, sensitivity, and reproducibility of the system for identifying and characterizing environmental estrogens.

• Journal of Plant Biotechnology
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• v.6 no.1
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• pp.15-19
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• 2004

The expression of a chimeric transgene (nos-npt II) has been examined in 9 years old transgenic poplars (Populus koreana x P. nigra) growing in a nursery. The expression of the gene in twenty six independentely transformed plants were examined by 1) enzyme (NPT II) assay, 2) RT-PCR, and 3) resistance to kanamycin. High NPT II activities in young leaves of all the transformed plants were found even without a selection pressure for antibiotics for 9 years. However, the activity varied with the positions of leaves in the stem in that young leaves showed higher activity than did mature tissues. When leaf segments were cultured in the presence of 150 mg/l kanamycin, only those from young leaves produced vigorously growing callus. However, as in the case of NPTII assay, the leaf segments from mature leaves did not form callus well on the media. RT-PCR with nptII specific primers also showed that amplification products were observed only when RNAs from young tissues were used. The total RNA gel showed that while RNA in young leaves are relatively stable and in a large quantity, those in old leaves were mostly degraded. All the above results suggest that the gene is transcriptionally active only in young tissue even though it is attached to a constituitive promoter. Therefore, the expression of foreign gene in poplar plants seemed to be affected by the metabolic state of the cells and thus vary greatly with the developmental stages and the age of tissue.

• Molecules and Cells
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• v.27 no.5
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• pp.577-582
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• 2009

The development of a high-throughput functional genomic screening provides a novel and expeditious approach in identifying critical genes involved in specific biological processes. Here we describe a cell-based cDNA screening system to identify the transcription activators of BiP, an endoplasmic reticulum (ER) chaperone protein. BiP promoter contains the ER stress element which is commonly present in the genes involved in unfolded protein response (UPR) that regulates protein secretion in cells. Therefore, the positive regulators of BiP may also be utilized to improve the recombinant protein production through modulation of UPR. Four BiP activators, including human UDP-glucose:glycoprotein glucosyltransferase 1 (HUGT1), are identified by the cDNA screening. Overexpression of HUGT1 leads to a significant increase in the production of recombinant erythropoietin, interferon ${\gamma}$, and monoclonal antibody in HEK293 cells. Our results demonstrate that the cDNA screening for BiP activators may be effective to identify the novel BiP regulators and HUGT1 may serve as an ideal target gene for improving the recombinant protein production in mammalian cells.

• KSBB Journal
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• v.23 no.5
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• pp.445-448
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• 2008

Candida antarctica lipase B (CalB) is an efficient biocatalyst for many organic synthesis reactions. To make full use of CalB, we need effective expression system. Previously recombinant CalB was successfully expressed in the methylotropic yeast Pichia pastoris. In addition, we succeed in the functional expression of CalB in the Escherichia coli cytoplasm. This CalB expression system in E.coli has many considerable advantages in comparison with other expression systems and enables high-throughput screening of gene libraries as those derived from directed evolution experiments. To optimize E.coli system, we investigate comparing between OrigamiB (DE3) and BL21 (DE3) and observing effect of IPTG amount.

• BMB Reports
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• v.49 no.8
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• pp.401-402
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• 2016

S6K1 is a key regulator of cell growth, cell size, and metabolism. Although the role of cytosolic S6K1 in cellular processes is well established, the function of S6K1 in the nucleus remains poorly understood. Our recent study has revealed that S6K1 is translocated into the nucleus upon adipogenic stimulus where it directly binds to and phosphorylates H2B at serine 36. Such phosphorylation promotes EZH2 recruitment and subsequent histone H3K27 trimethylation on the promoter of its target genes including Wnt6, Wnt10a, and Wnt10b, leading to repression of their expression. S6K1-mediated suppression of Wnt genes facilitates adipogenic differentiation through the expression of adipogenic transcription factors PPARγ and Cebpa. White adipose tissues from S6K1-deficient mice consistently exhibit marked reduction in H2BS36 phosphorylation (H2BS36p) and H3K27 trimethylation (H3K27me3), leading to enhanced expression of Wnt genes. In addition, expression levels of H2BS36p and H3K27me3 are highly elevated in white adipose tissues from mice fed on high-fat diet or from obese humans. These findings describe a novel role of S6K1 as a transcriptional regulator controlling an epigenetic network initiated by phosphorylation of H2B and trimethylation of H3, thus shutting off Wnt gene expression in early adipogenesis.

• Environmental Mutagens and Carcinogens
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• v.25 no.1
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• pp.40-46
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• 2005

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) displays high toxicity in animals and has been implicated in human carcinogenesis. Although the mechanism of carcinogenesis by TCDD is unclear, it is considered to be a non-genotoxic compound and tumor promoter. In our experiment, we investigated the effects of TCDD on gene expression in mouse skin carcinogenesis. We used cDNA microarray to detect the differential gene expression in tumors induced in hairless mouse skin by MNNG plus TCDD protocol. We found that erb-2, c-ets2 and p27$^{kip1}$ were significantly up-regulated, but TNFR2, AKT-l, integrin $\beta$l, maspin, IGF-l, c-raf-l, Rb were significantly down-regulated, in tumor region, respectively. We also found that the expression of 53 genes involved in cen cycle, signal transduction, apoptosis, adhesion molecule, angiogenesis, and invasion, were changed two fold more, in tumor surrounding region. These data suggest that TCDD alters the expression of a large array of genes involved in apoptosis, cytokine production and angiogenesis in mouse skin carcinogenesis.