• The Korean Journal of Food And Nutrition
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• v.9 no.4
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• pp.452-458
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• 1996

A PPIase gene of Bacillus stearothermophilus was screened from a genomic library by plaque hybridization using the A-1 primer as a probe. A PPIase positive plaque contained a 3.0kb insert of the chromosomal DNA. A 3.0kb fragment was subcloned into pUC18, resulting pPI1-40. A DNA fragment encoding the N-terminal portion of the PPIase in pPi-40 was amplified by polymerase chain reaction(PCR) method using the A-1 and B-2 primers. The amplified fragment was cloned into the Sma I site of pUC18 and recombinant plasmid was designated as pSN-18. The nucleotide sequence of 167bp fragment was determined. The deduced amino acid sequence of PPIase was completely matched with the determined N-terminal amino acid sequence of PPIase B. stearothermophilus. The translated protein sequence of PPIase B. stearothermophilus was compared with sequence from periplasmic PPIase from Escherichina coil ; homogies of 16 and 58%, respectively, were found. The clond PPIase gene was over-expressed in E. coil cell using pUC19 as an expression vector. The enzyme was partially purified by heat treatment and colum chromatochraphy on DEAE-Sepharose CL-6B. The molecular weight of the enzyme was dermined to be about 18.0 kDal by SDS-PAGE.

• Journal of The Korean Society of Grassland and Forage Science
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• v.17 no.4
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• pp.379-386
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• 1997

Recombinant plasmid, pBKH4, containing NPT II and P35S-BcHSP17.6 was constructed by ligation of Bum H I -digested pBKSl-l and BcHSP 17.6 (thermotolerance gene) 6om pBLH4. The tobacco leaf disc was cocultivated with transformed Agmbacterium tumefaciens bearing pBKH4 for 24 hours and transformed shoots were selected on MS-n/B medium containing $100\;{\mu\textrm{g}}/ml$ of kanamycin. Heat-killing temperature of Nicotima tabacum was $50^{\circ}$ for >15min, and transformed tobacco plants with BcHSP17.6 cDNA exhibited thermotolerance at the heat-killing temperature. The transgenic plants were analyzed by Southern blot hybridization with the probe of ${\alpha}^{_32}P$ labelled BcHSP17.6 cDNA. Transcription and expression level of BcHSP17.6 cDNA were also continued by Northern blot analysis and Ouchterlony double immunodiffusion assay. In this study, we suggest that the BcHSP17.6 cDNA introduced to tobacco plant is related to thenuoto-lerance and 17.6-kD LMW HSP acts as a protector from heat damage in plants.

• Journal of Life Science
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• v.25 no.5
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• pp.507-514
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• 2015

D-Xylulose is phosphorylated to D-xylulose-5-phosphate by D-xylulose kinase before it enters glycolysis via the nonoxidative pentose phosphate pathway. A gene encoding a novel D-xylulose kinase (XK) from K. gwangalliensis strain SJ2 was sequenced and expressed in E. coli. The sequence of the isolated XK gene was 1,419 bp, encoding 472 amino acids. The XK protein was more closely related to the Arthrobacter phenanthrenivorans XK than to the Bifidobacterium catenulatum one, as reflected in the sequence identity (54.9% vs. 38.7%). The XK gene was subcloned into the pCold-II expression vector. The resulting plasmid was transformed into E. coli strain BL21 (DE3) cells and the expression of the recombinant XK protein was induced by the addition of IPTG. The resulting protein was expressed as a fusion protein of approximately 48 kDa containing a N-terminal six-histidine extension that was derived from the expression vector. The expressed protein was homogenized by affinity chromatography and showed enzymatic activity corresponding to D-xylulose kinase. XK enzyme kinetic studies with D-xylulose and ATP showed a Km of 250±20 μM and 1,300±50 μM, respectively. The results obtained from this study will provide a wider knowledge base for the characterization of D-xylulose kinase at the molecular level.

• Korean Journal of Microbiology
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• v.52 no.1
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• pp.1-9
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• 2016

A unique Schizosaccharomyces pombe $TrxR^+$ gene encoding thioredoxin reductase (TrxR) was found to be positively regulated by stress-inducing agents through the stress-responsive transcription factor Pap1. In the present study, the protective roles of S. pombe TrxR were evaluated using the TrxR-overexpressing recombinant plasmid pHSM10. In the presence of hydrogen peroxide ($H_2O_2$) and superoxide anion-generating menadione (MD), S. pombe TrxR increased cellular growth and the total glutathione (GSH) level, while it reduced levels of intracellular reactive oxygen species (ROS). The nitric oxide (NO) levels of the TrxR-overexpressing cells, in the presence of $H_2O_2$ and MD, were maintained to be similar to those of the corresponding non-treated cells. Although S. pombe TrxR was able to scavenge NO generated by sodium nitroprusside (SNP), it had no significant modulating effects on cellular growth, ROS levels, or the total GSH level of SNP-exposed yeast cells, compared with the differences in those of the two non-treated cell cultures. TrxR increased the cellular growth and total GSH level, which were diminished by nitrogen starvation. It also scavenged ROS and NO produced during nitrogen starvation. Taken together, the S. pombe TrxR protects against oxidative, nitrosative, and nutritional stresses.

• Journal of Microbiology and Biotechnology
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• v.26 no.11
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• pp.1881-1890
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• 2016

Manganese superoxide dismutase (MnSOD) is a vital enzyme that protects cells from free radicals through eliminating superoxide radicals ($O^{2-}$). Hirudin, a kind of small active peptide molecule, is one of the strongest anticoagulants that can effectively cure thrombus diseases. In this study, we fused Hirudin to the C terminus of human MnSOD with the GGGGS linker to generate a novel dual-feature fusion protein, denoted as hMnSOD-Hirudin. The hMnSOD-Hirudin gene fragment was cloned into the pET15b (SmaI, CIAP) vector, forming a recombinant pET15b-hMnSOD-Hirudin plasmid, and then was transferred into Escherichia coli strain Rosetta-gami for expression. SDS-PAGE was used to detect the fusion protein, which was expected to be about 30 kDa upon IPTG induction. Furthermore, the hMnSOD-Hirudin protein was heavily detected as a soluble form in the supernatant. The purification rate observed after Ni NTA affinity chromatography was above 95%. The hMnSOD-Hirudin protein yield reached 67.25 mg per liter of bacterial culture. The identity of the purified protein was confirmed by western blotting. The hMnSOD-Hirudin protein activity assay evinced that the antioxidation activity of the hMnSOD-Hirudin protein obtained was $2,444.0{\pm}96.0U/mg$, and the anticoagulant activity of the hMnSOD-Hirudin protein was $599.0{\pm}35.0ATU/mg$. In addition, in vitro bioactivity assay showed that the hMnSOD-Hirudin protein had no or little cytotoxicity in H9c2, HK-2, and H9 (human $CD_4{^+}$, T cell) cell lines. Transwell migration assay and invasion assay showed that the hMnSOD-Hirudin protein could suppress human lung cancer 95-D cell metastasis and invasion in vitro.

• Korean Journal of Plant Tissue Culture
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• v.24 no.5
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• pp.305-311
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• 1997

Bacillus thuringiensis, a gram-positive soil bacterium, is characterized by its ability to produce crystalline inclusions during sporulation. The crystal proteins exhibit a highly specific insecticidal activity. An insecticidal crystal protein (ICP), Cry II A, is specifically toxic to both lepidopteran and dipteran insects. In this study, tobacco plants transformed by the cry II A gene have been generated. The Cry II A crystal protein was purified from E. coli JM103 harboring cry II A gene by differential solubility. The activated Cry II A was prepared by tryptic digestion. The purified protoxin (70 kDa) and the activated toxin (50 kDa) were analyzed by SDS-PAGE. To generate the transgenic tobacco having cry II A gene, the cry II A gene was subcloned to a plant expression vector, pSRL2, having two CaMV 35S promoters. The recombinant plasmid was transformed into tobacco (N. tabacum var. Petit Havana SR1) by Agrobacterium-mediated leaf disc transformation. Through the regeneration, six putative transgenic tobacco plants were obtained and three transformants were confirmed by Southern blot analysis. It has been found that one plant had single copy of cry II A gene, another had two copies of the gene, and the third had a truncated gene. After the immunochemical confirmation of cry II A expression in plants, the transgenic tobacco plants will be used to study the genetics of future generation with the insecticidal crystal protein gene cry II A.

• Korean Journal of Microbiology
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• v.45 no.4
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• pp.419-424
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• 2009

To provide the basis of biosensor based on the lux genes from bioluminescent bacteria of Photobacterium leiognathi and Vibrio harveyi, we test the expression of lux genes in several strains of Escherichia coli. The expression of the recombinant plasmid of PlXba.pT7-3, containing all lux genes requiring for light emission without adding substrate, in E. coli 43R was so strong to see the blue-green light in single colony as well as in the alginate immobilized cell. In addition, the light intensity was decreased by adding heavy metal ion such as cadmium and zinc ions. These result raise the possibility that a biosensor can be developed using the lux genes system.

• Korean Journal of Microbiology
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• v.36 no.3
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• pp.196-202
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• 2000

A gene for cellulolytic xylanase of Bacillus circulnns ATCC21365 was cloned on pUC 19 in Eschwichia coli. The recombinant plasniid pXLI80 contained an 1.8 id, inselt composed of0.5 kb and 1.3 kb PslI fragments derived from B, circulans. The 0.5 kh fragment in the upstream region of 1.3 kb one was confirmed lo be indispensable for not only expression but also hyperexpression of the cloned gene. The transformant overproduced the xylanase 135 times greater than that produced by the orlginal B circulnns. The optimum pH and temperature of the cloned enzyme we]-e pH 5.2 and $60^{\circ}C$, respectively. Heal pretl-eatment at TEX>$55^{\circ}C$C for 1 Indid not cause inhibition of the activity of this enzyme. The elm.ynie could hydl-olyre CMC and lichenan as well as xylan to produce xylose(or GI), xylohiose(or G2) and xylolnose(or G3) as inah products. Hence We defined the cloned enzyme as a cellulolytic xylanase. The SDS-PAG electrophoretic mobility and zyiiogram of this enzyme derived from whole cell extracts or c~~lture supematants or E. coli(pXL180) indicated a molecular weight of 45,000 and nonprocessing of the enzyme in the peilplasln of E. coli.

• Proceedings of the Korean Society of Life Science Conference
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• 2001.06a
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• pp.67-86
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• 2001

A strain producing strongly fibrinolytic enzyme was isolated from soil and was identified to be Bacillus subtilis by biochemical and physiological characterization. The optimal culture conditions for the production of fibrinolytic enzyme was determined to be 1.0% tryptone, 1.5% soluble starch, 0.5% Peptone, 0.5% NaCl, $(NH_{4})_{3}PO_4.3H_{2}O, and MgSO_{4}.7H_{2}O.$ Initial pH and temperature were pH 8.0 and $30^{\circ}C$ , respectively, The highest enzyme production was observed at 30 hours of cultivation at $30^{\circ}C$ The fibrinolytic enzyme was purified to homogeneity by DEAE Sephadex A-50 ion exchange column chromatography, 70% ammonium sulfate precipitation, Sephadex G-200 and G-75 gel filtration column chromatography. The molecular weight of the purified enzyme was 28,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A gene encoding the fibrinolytic enzyme was cloned into a plasmid vector pBluescript, transforming E.coli XL-1 Blue. The clone was able to degrade fibrin, This indicated that the gene could encode a fibrinolytic enzyme. The nucleotide sequence of the 2.7 kb insert was determined in both direction. One open reading frame composed of 1023 nucleotides was found to be a potential protein coding region. There was the putative Shine-Dalgano sequence and TATA box upstream of the open reading frame. The homology search data in the genome database showed that both the 2.7 kb insert and 1 kb open reading frame carried no significance in the nucleotide sequence of known fibrinolytic enzyme from Bacillus serovars. The recombinant cell harboring the novel gene involved in fibrinolysis was subjected to protein purification. The molecular mass of the purified fibrinolytic enzyme was determined to be 31864 Dalton, which was highly in accordance with the molecular mass(33 kDa) of the fibrinolytic gene deduced from the insert. The fibrinolytic enzyme was Purified 50.5 folds to homogeneity in overall yield of 10.7% by DEAE Sephadex A-50 ion exchange, 85% ammonium sulfate precipitation, Sephadex G-50, Superdex 75 HR FPLC gel filtration. In conclusion, a novel fibrinolytic gene from Bacillus subtilis was identified and characterized by cloning a genomic library of Bacillus subtilis into pBleuscript. For the soybean fermented by this strain, it is found that there increased assistant protein about 20% compared to the soybean not fermented and increased about 30% according to amino acid analysis and, in particular, essential amino acid increased about 40%. When keeping this fermented soybean powder at room temperature for about 70days, it showed very high stability maintaining almost perfect activity and, therefore, it gave us great suggestion its possibility of development as a new functional food.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.916-922
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• 2005

Parkin, known as an E3 ubiquitin ligase, has essential role in protein quality control, and its severe dysfunction leads to neurodegenerative disorders. Human Parkin was excessively degraded when expressed in Escherichia coli under the conventional induction condition ($37^{\circ}C$ culture condition with 0.5 mM IPTG). To optimize the induction and culture conditions for recombinant human Parkin and develop a rapid method for the Parkin purification, we expressed Parkin by using PCEX system at the different culture temperatures and IPTC concentrations. The intact Parkin protein was purified to approximately $90\%$ purity with suitable amounts of protein under the optimal culture condition ($25^{\circ}C$E with 0.01 mM IPTG). Additionally, we constructed various parkin plasmids with different tagging systems and investigated their expression patterns in HEK293 cells. We found that the proteolytically sensitive site is localized within a ubiquitin-like domain of Parkin. This study developes a method for generating useful reagents to investigate biochemical properties of Parkin.