• KSBB Journal
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• v.30 no.6
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• pp.291-295
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• 2015

The limited productivity of natural shell matrix proteins has hampered the investigation of their biochemical properties and practical applications, although biominerals in nature obtained by organic-inorganic assemblies have attractive mechanical and biological properties. Here, we prepared a vector for the expression of a fusion protein of a shell matrix protein from Pinctada fucata (named as GRP_BA) with the GRGDSP residue. The fusion protein of BA-RGD was simply produced in E. coli and purified through sequential steps including the treatment with $CaCl_2$ and EDTA solution for cell membrane washing, mechanical cell disruption and the application of non-ionic surfactant of Triton X-100 for BA-RGD inclusion body washing. The production yield was approximately 60 mg/L, any other protein band was not observed in SDS-PAGE and it was estimated that above 97% endotoxin was removed compared to the endotoxin level of whole cell. This study showed this simple and easy purification approach could be applied to the purification of BA-RGD fusion protein. It is expected that the protein could be utilized for the preparation of biominerals in practical aspects.

• BMB Reports
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• v.32 no.4
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• pp.363-369
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• 1999

The orf8(chlD) gene cloned from Streptomyces antibioticus T$\"{u}$99 was overexpressed using an E. coli system to confirm its biological function. Induction of the E. coli strain transformed with recombinant plasmid pRFJ 1031 containing orf8 resulted in the production of a 43,000 dalton protein. Glucose-1-phosphate thymidylyltransferase activity of the cell extract obtained from the transformed strain was 4-5 times higher than that of the control strain. The expressed protein was purified 18-fold from E. coli cell lysate using three chromatographic steps with a 17% overall recovery to near homogeneity. The N-terminal amino acid sequence of the purified protein agrees with the nucleotide sequence predicted from the orf8 gene. The SDS-PAGE estimated subunit mass of 43,000 dalton agrees well with that calculated from the amino acid composition deduced from the nucleotide sequence of the orf8 gene (43,000 Da). Also, the native enzyme has a monomeric structure with a molecular mass of 43,000 dalton. The purified protein showed glucose-1-phosphate thymidylyltransferase activity catalyzing a reversible bimolecular group transfer reaction, and was highly specific for dTTP and ${\alpha}$-D-glucose 1-phosphate as substrates in the forward reaction, and for dTDP-D-glucose and pyrophosphate in the reverse reaction.

• BMB Reports
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• v.43 no.8
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• pp.573-578
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• 2010

Several organophosphorus (OP) insecticides can selectively kill the silkworm maggot, Exorista sorbillans (Es) (Diptera: Tachinidae), while not obviously affecting the host (Bombyx mori) larvae, but the mechanism is not yet clear. In this study, the cDNA encoding an acetylcholinesterase (AChE) from the field Es was isolated. One point mutation (Gly353Ala) was identified. The Es-353G AChE and Es-353A AChE were expressed in baculovirus-insect cell system, respectively. The inhibition results showed that for eserine and Chlorpyrifos, Es-353A AChE was significantly less sensitive than Es-353G AChE. Meanwhile, comparison of the I50 values of eserine, dichlorvos, Chlorpyrifos and omethoate of recombinant Es AChEs with its host (Bombyx mori) AChEs indicated that, both Es AChEs are more sensitive than B. mori AChEs. The results give an insight of the mechanism that some OP insecticides can selectively kills Es while without distinct effect on its host, B. mori.

• Molecules and Cells
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• v.23 no.1
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• pp.72-79
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• 2007

Demand for low-input sustainable crop cultivation is increasing to meet the need for environment-friendly agriculture. Consequently, developing genotypes with high nutrient use efficiency is one of the major objectives of crop breeding programs. This study was conducted to identify QTLs for traits associated with physiological nitrogen use efficiency (PNUE). A recombinant inbred population (DT-RILs) between Dasanbyeo (a tongil type rice, derived from an indica ${\times}$ japonica cross and similar to indica in its genetic make-up) and TR22183 (a Chinese japonica variety) consisting of 166 $F_8$ lines was developed and used for mapping. A frame map of 1,409 cM containing 113 SSR and 103 STS markers with an average interval of 6.5 cM between adjacent marker loci was constructed using the DT-RILs. The RILs were cultivated in ordinary-N ($N-P_2O_5-K_2O=100-80-80kg/ha$) and low-N ($N-P_2O_5-K_2O=50-80-80kg/ha$) (100 kg/ha) conditions. PNUE was positively correlated with the harvest index and grain yield in both conditions. Twenty single QTLs (S-QTLs) and 58 pairs of epistatic loci (E-QTLs) were identified for the nitrogen concentration of grain, nitrogen concentration of straw, nitrogen content of shoot, harvest index, grain yield, straw yield and PNUE in both conditions. The phenotypic variance explained by these S-QTLs and E-QTLs ranged from 11.1 to 44.3% and from 16.0% to 63.6%, respectively. The total phenotypic variance explained by all the QTLs for each trait ranged from 35.8% to 71.3%, showing that the expression of PNUE and related characters depends signify- cantly upon genetic factors. Both S-QTLs and E-QTLs may be useful for marker-assisted selection (MAS) to develop higher PNUE genotypes.

• Molecules and Cells
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• v.23 no.1
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• pp.17-22
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• 2007

We have expressed human erythropoietin (EPO) in transgenic mice using a recombinant EPO cDNA combined with a partial TPO construct. The gene was microinjected using standard techniques and five mice were detected as transgenic by PCR and further used as founders. The life span of the transgenic founders was much shorter than that of their normal littermates. Most of the tissues of the transgenic founders contained human EPO transcripts as judged by RT-PCR. Especially high expression levels were seen in the liver and lung. EPO protein levels in serum were examined by ELISA and ranged from 266-414 mIU/ml. The number of red blood cell, white blood cell and hemoglobin in the hEPO transgenic mice was higher than in normal mice. These results indicate that overexpression of hEPO is deleterious and can provoke lung failure and erythrocytosis.

• Journal of Embryo Transfer
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• v.25 no.1
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• pp.35-42
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• 2010

Many studies propose that dysfunction of mitochondrial proton-translocating NADH-ubiquinone oxidoreductase (complex I) is associated with neurodegenerative disorders, such as Parkinson's disease and Huntington's disease. Mammalian mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I) consists of at least 46 different subunits. In contrast, the NDI1 gene of Saccharomyces cerevisiae is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. With a recombinant adeno-associated virus vector carrying the NDI1 gene (rAAV-NDI1) as the gene delivery method, we were able to attain high transduction efficiencies even in the human epithelial cervical cancer cells that are difficult to transfect by lipofection or calcium phosphate precipitation methods. Using a rAAV-NDI1, we demonstrated that the Ndi1 enzyme is successfully expressed in HeLa cells. The expressed Ndi1 enzyme was recognized to be localized in mitochondria by confocal immunofluorescence microscopic analyses and immunoblotting. Using digitonin-permeabilized cells, it was shown that the NADH oxidase activity of the NDI1-transduced HeLa cells were not affected by rotenone which is inhibitor of complex I, but was inhibited by flavone and antimycin A. The NDI1-transduced cells were able to grow in media containing rotenone. In contrast, control cells that did not receive the NDI1 gene failed to survive. In particular, in the NDI1-transduced cells, the yeast enzyme becomes integrated into the human respiratory chain. It is concluded that the NDI1 gene provides a potentially useful tool for gene therapy of mitochondrial diseases caused by complex I deficiency.

• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.797-804
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• 2000

The human immunodeficiency virus type 1 (HIV-1) Tat is one of the viral gene products essential for HIV replication. The exogenous Tat protein is transduced through the plasma membrane and then accumulated in a cell. The basic domain of the Tat protein, which is rich in arginine and lysine residues and called the protein transduction domain (PTD), has been identified to be responsible for this transduction activity. To better understand the nature of the transduction mediated by this highly basic domain of HIV-1 Tat, the Green Fluorescent Protein (GFP) was expressed and purified as a fusion protein with a peptide derived from the HIV-1 Tat basic domain in Escherichia coli. The transduction of Tat-GFP into mammalian cells was then determined by a Western blot analysis and fluorescence microscopy. The cells treated with Tat-GFP exhibited dose- and time-dependent increases in their intracellular level of the protein. the effective transduction of denatured Tat-GFP into both the nucleus and the cytoplasm of mammalian cells was also demonstrated, thereby indicating that the unfolding of the transduced protein is required for efficient transduction. Accordingly, the availability of recombinant Tat-GFP can facilitate the simple and specific identification of the protein transduction mediated by the HIV-1 Tat basic domain in living cells either by fluorescence microscopy or by a fluorescence-activated cell sorter analysis.

• Biomolecules & Therapeutics
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• v.28 no.2
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• pp.152-162
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• 2020

Cerebral ischemia exhibits a multiplicity of pathophysiological mechanisms. During ischemic stroke, the reactive oxygen species (ROS) concentration rises to a peak during reperfusion, possibly underlying neuronal death. Recombinant human erythropoietin (EPO) supplementation is one method of treating neurodegenerative disease by reducing the generation of ROS. We investigated the therapeutic effect of PEGylated EPO (P-EPO) on ischemic stroke. Mice were administered P-EPO (5,000 U/kg) via intravenous injection, and middle cerebral artery occlusion (MCAO) followed by reperfusion was performed to induce in vivo ischemic stroke. P-EPO ameliorated MCAO-induced neurological deficit and reduced behavioral disorder and the infarct area. Moreover, lipid peroxidation, expression of inflammatory proteins (cyclooxygenase-2 and inducible nitric oxide synthase), and cytokine levels in blood were reduced by the P-EPO treatment. In addition, higher activation of nuclear factor kappa B (NF-κB) was found in the brain after MCAO, but NF-κB activation was reduced in the P-EPO-injected group. Treatment with the NF-κB inhibitor PS-1145 (5 mg/kg) abolished the P-EPO-induced reduction of infarct volume, neuronal death, neuroinflammation, and oxidative stress. Moreover, P-EPO was more effective than EPO (5,000 U/kg) and similar to a tissue plasminogen activator (10 mg/kg). An in vitro study revealed that P-EPO (25, 50, and 100 U/mL) treatment protected against rotenone (100 nM)-induced neuronal loss, neuroinflammation, oxidative stress, and NF-κB activity. These results indicate that the administration of P-EPO exerted neuroprotective effects on cerebral ischemia damage through anti-oxidant and anti-inflammatory properties by inhibiting NF-κB activation.

• Biomolecules & Therapeutics
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• v.2 no.3
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• pp.286-291
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• 1994

The mutagenicity of DA-3030(rhG-CSF)was studied by reverse mutation test, chromosome aberration test and micronucleus test. The reverse mutatuon test in bacteria was performed using salmonella typhimurium strain TA100, TA98, TA1535 and TA1537 with rhG-CSF in any of the concentrations(150, 75, 37.5, 18.75, 9.375 and 4,6875 $\mu\textrm{g}$/plate), no increase in the number of revertant colonies in each strain was observed, irrespective of treatment with the metabolic activation system(S-9 mix) The chromosome aberration test was carried out using CHL cells, cell line from chinese hamster lung. With 4 doses(75, 37.5, 18.75 and 9.375 $\mu\textrm{g}$/ml) of rhG-/CSF the cells were treated for 24 or 48 hours in the direct method or for 6 hours followed by 18 hour-expression time in the metabolic activation method. Results of the study showed, by the direct method or metabolic activation method, no trend toward increase in the number of aberrant metaphase. The micronucleus test was carried out using ICR mice at the age of 8 weeks. Three doses(862.5, 1725 and 3450 $\mu\textrm{g}$/kg) of DA-3030 were admintstered intraperitoneally with single shot and bone marrow cells were sampled at 24 hours after administration. Neither the number of polychromatic erythrocytes with micronuclei nor the ratio of normochromatic erythrocytes to polychromatic erythrocytes increased singinficantly in each dose, compared with a vehicle control. These results indicate that rhG-CSF has not mutagenic potential under the condiions.

• Bulletin of the Korean Chemical Society
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• v.32 no.6
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• pp.1925-1930
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• 2011

Psychrophilic bacteria have acquired cold-resistance in order to protect themselves against freezing temperatures, which would otherwise be lethal. DnaK/DnaJ/GrpE systems are molecular chaperones which facilitate proper folding of newly synthesized proteins. Efficient folding processes are of great importance especially in a cold environment, such as the Arctic. In order to understand the protection mechanisms of psychrophilic bacteria against cold temperatures, we have explored a genome of KOPRI22215, tentatively identified as Psychromonas arctica, whose genome sequence has not yet been discovered. With an aim of searching for a coding gene of DnaK from KOPRI22215, we have applied a series of polymerase chain reactions (PCR) with homologous primers designed from other Psychromonas species and LA PCR in vitro cloning. 1917 bp complete coding sequence of dnaK from KOPRI22215 was identified including upstream promoter sites. Recombinant plasmids to overexpress PaDnaK along with EcDnaK (DnaK of E. coli) were then constructed in pAED4 vector and the pET-based system to induce PaDnaK expression by IPTG. Characterization assays of expressed PaDnaK were carried out by measuring survival rates upon 4 day incubation at 4 $^{\circ}C$: a refolding assay as molecular chaperone, and ATPase assay for functional activity. Taking account of all the data together, we conclude that PaDnaK was identified, successfully expressed, and found to be more efficient in providing cold-resistance for bacterial cells.