• KSBB Journal
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• v.21 no.3
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• pp.204-211
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• 2006

For the production of the recombinant human interferon-gamma(rhIFN-${\gamma}$) in Escherichia coli, human glucagon and ferritin heavy chain were used as fusion partners. Even though rhIFN-${\gamma}$ is expressed as an inclusion body form in E. coli because of strong hydrophobicity of itself, over 50% of fused rhIFN-${\gamma}$ was expressed as soluble form in E. coli $Origami^{TM}$(DE3) harboring pT7FH(HE)-IFN-${\gamma}$ which encodes ferritin heavy chain-fused rhIFN-${\gamma}$. In the case of using glucagon-ferritin heavy chain hybrid mutant as a fusion partner, 6X His-tag was additionally introduced to N-terminus of GFHM(HE)-IFN-${\gamma}$ for enhancing purification yields of rhIFN-${\gamma}$. Fusion protein HGFHM(HE)-IFN-${\gamma}$ with two 6X His-tag was more effectively bound to Ni-NTA agarose bead than GFHM(HE)-IFN-${\gamma}$ with a 6X His-tag. rhIFN-${\gamma}$ was completely purified from enterokinase-treated HGFHM(HE)-IFN-${\gamma}$ by Ni-NTA affinity column. For high-level production of rhIFN-${\gamma}$, glucose was used as the sole carbon source with simple exponential feeding rate($2.4{\sim}7.2g/h$) in fed-batch process. The effective lactose concentration for the expression of the rhIFN-${\gamma}$ was $10{\sim}20mM$. Under the fed-batch culture conditions, rhIFN-${\gamma}$ production yield reached 11 g DCW/L for 6 hours after lactose induction.

• Food Science and Preservation
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• v.31 no.3
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• pp.423-432
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• 2024

In this study, a vacuum tumbler with 4 impellers (DVT) was designed and applied for thawing frozen pork (vacuum -60 kPa, jacket 35℃, 1 rpm). Quality characteristics of the thawed pork were compared with those of industrially thawed meat by natural air at room temperature (NAT) and imported vacuum tumbler (IVT). The thawing time for frozen pork (303.36 kg) using DVT (165 min) was much shorter than that of NAT (4,200 min). DVT-thawed pork had lower drip loss (0.85%) than NAT (2.08%). DVT-thawed pork showed a pH of 5.92, a total bacterial count of 1.96±0.02 log CFU/g and no coliforms. Deteriorations in fat (TBARS 0.31±0.01 MDA mg/kg) and protein (VBN 5.67±1.98 mg%) in DVT-thawed pork were significantly lower than those of NAT (p<0.05). DVT-thawed pork had a high water-holding capacity (WHC, 97.5%). The hardness (34.59±0.46 N) and chewiness (188.21±0.17) of cooked DVT-thawed pork were about 5-6 times lower than those of NTA. Microstructure (SEM) showed myofibrillar damage in NAT-thawed pork, whereas dense myofibrillar structure was observed in DVT-thawed pork. DVT was better or similar to IVT in all evaluation parameters. The designed DVT is expected to be used as an efficient thawing method in terms of processing time and yield and to produce thawed meat with high WHC, soft texture, and low spoilage by minimizing tissue damage.

• Nuclear Engineering and Technology
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• v.16 no.2
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• pp.58-63
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• 1984

Paper electrophoretic separation of octahedrally bonded (Ruc $l_{6}$ )$^{3-}$ has been carried out by using the specially designed migration apparatus. The supporting electrolyte solutions are as follows: 0.1M-HCl $O_4$, 0.05 M-HCl+0.09M-KCl, 0.1M-HCl, 5$\times$10$^{-3}$ M-NTA, 0.01M-HCl, 0.01M-HCl $O_4$, 0.01M-citric acid, 0.01M-K $H_2$P $O_4$+0.01M-N $a_2$HP $O_4$, 0.05M-borax, 0.025M-N $a_2$C $O_3$+0.025M-NaHC $O_3$, 0.01M-N $a_3$P $O_4$, 0.01M-NaOH and 0.1 M-NaOH. The (Ruc $l_{6}$ )$^{3-}$ appears in 2 to 4 peaks and is found in several chemical species such as (RuCl ($H_2O$)$_{5}$ )$^{2+}$, cis and trans (RuC $l_2$($H_2O$)$_4$)$^{1+}$, (RuC $l_3$($H_2O$)$_3$)$^{0}$ , (RuC $l_4$($H_2O$)$_2$)$^{1-}$, (RuC $l_{5}$ ($H_2O$))$^{2-}$ and (RuC $l_{6}$ )$^{3-}$. The retention value has been found to be highest in the 0.025M-N $a_2$C $O_3$+0.025M-NaHC $O_3$ electrolyte solution.n.

• Tuberculosis and Respiratory Diseases
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• v.58 no.2
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• pp.142-152
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• 2005

Background : Priming and boosting vaccination strategy has been widely explored for new vaccine development against tuberculosis. As an effort to identify other vaccine candidates, this study was initiated to evaluate protective efficacy of adenylate kinase (AK), nucleoside diphosphate kinase (NdK), and heat shock protein 70 (Hsp70) of Mycobacterium tuberculosis. Method : M. tuberculosis genes encoding AK, NdK, and Hsp70 proteins were amplified by PCR and cloned into E. coli expression vector, pQE30. Recombinant AK, NdK, and Hsp70 was purified through Ni-NTA resin. To evaluate immune responses, we performed enzyme-linked immunosorbent assay (ELISA) for IgG isotype and $IFN-{\gamma}$ after mice were immunized subcutaneously with recombinant proteins delivered in dimethyl dioctadecylammonium bromide (DDA). Immunized- and control groups were challenged by aerosol with M. tuberculosis. The spleens and lungs of mice were removed aseptically and cultured for CFU of M. tuberculosis. Result : Vaccination with recombinant proteins AK, NdK, and Hsp70 delivered in DDA elicited significant level of antibody and $IFN-{\gamma}$ responses to corresponding antigens but no protective immunity comparable to that achieved with Mycobacterium bovis BCG. Conclusion : Recombinant proteins AK, NdK, and Hsp70 do not effectively control growth of M. tuberculosis in mice when immunized with DDA as an adjuvant.

• Korean Journal of Environmental Agriculture
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• v.24 no.4
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• pp.370-378
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• 2005

The characteristics of heavy metal biosorption on the seaweeds were investigated to develop a biological treatment technology for wastewater polluted with heavy metals. The heavy metal biosorption on seaweeds ranked in the tallowing order: U. pinnatifida$\geq$E. stolonifera$\geq$Laminaria sp.>G. amansii. The Pb was biosorbed in the range of $93{\sim}99%$, and the Cu and Cd were biosorbed in the range of $70{\sim}80%$ at the concentration of the heavy metal of $100mg/{\ell}$ respectively. The seaweed which was pretreated with $CaCl_2$ solution improved the biosorption of the heavy metals. The temperature and pH didn't affect the biosorption of heavy metals. The Langmuir isotherm reasonably fit the data of heavy metal biosorption compared to the Freundlich isotherm. The affinity of metals on the biosorption ranked in the following order: Pb>Zn>Cu>Cd. The biosorption efficiency of the heavy metals on the U. pinnatifida decreased in the multi-component rather than the single component. The heavy metals adsorbed on the U. pinnatifida were recovered using 0.3%-NTA. U. pinnatifida among the seaweed used in this work showed the best performance for the biosorption of the heavy metals.

• Journal of Sericultural and Entomological Science
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• v.52 no.1
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• pp.45-51
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• 2014

A cecropin-A gene was isolated from the immunized larvae of the Japanese oak silkworm, Antheraea yamamai and designed Ay-CecA. The complete Ay-CecA cDNA consists of 419 nucleotides with 195 bp open reading frame encoding a 64 amino acid precursor that contains a putative 22-residue signal peptide, a 4-residue propetide and a 37-residue mature peptide with a theoretical mass of 4046.81. The deduced amino acid sequence of the peptide evidenced a significant degree of identity (62 ~ 78% identity) with other lepidopteran cecropins. Like many insect cecropin, Ay-CecA also harbored a glycine residue for C-terminal amidation at the C-end, which suggests potential amidation. To understand this peptide better, we successfully expressed bioactive recombinant Ay-CecA in Escherichia coli that are highly sensitive to the mature peptide. For this, we fused mature Ay-CecA gene with insoluble protein ketosteroid isomerase (KSI) gene to avoid the cell death during induction. The fusion KSI-CecA protein was expressed as inclusion body. The expressed fusion protein was purified by Ni-NTA immobilized metal affinity chromatography (IMAC), and cleaved by cyanogen bromide (CNBr) to release recombinant Ay-CecA. The purified recombinant Ay-CecA showed considerably antibacterial activity against Gram-negative bacteria, E. cori ML 35, Klebsiella pneumonia and Pseudomonas aeruginosa. Our results proved that this peptide with a potent antibacterial activity may play a role in the immune response of Japanese oak silkworm.

• Applied Biological Chemistry
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• v.46 no.4
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• pp.353-360
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• 2003

For the simple rapid bioassay of organophosphorus and carbamate pesticide residues, a mass-production system of acetycholinesterase (AChE, EC 3.1.1.7, MAChE) using baculovirus and insect cell culture was constructed. The cDNA for AChE was synthesized from Drosophila melanogaster in Halla Mountain, the lipid anchor tail was removed by PCR and was used for the site-directed mutagenesis of three amino acid residues (E107Y, F368L, L408F). The mutated cDNA was inserted into the baculovirus vector and expressed in insect cells. Maximum cell growth and enzyme activity were reached when the cells $(2{\times}10^6\;cell/ml)$ were infected four times at four-day-intervals. His-tag containing MAChE was purified using Ni-NTA column and used for characterization. The activity was maintained under various pHs (3-10) and temperatures $(20-50^{\circ}C)$ under experimental conditions. As an extraction solution for pesticides, methanol is more effective than ethanol. Against major organophosphate and carbamate pesticides, the MAChE showed better sensitivity than AChE and AChE from housefly (Taiwan).

• Applied Biological Chemistry
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• v.38 no.1
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• pp.49-54
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• 1995

To understand the molecular structure and pathogenesis mechanism of Korean garlic viruses, we have isolated cDNA clones for garlic viruses. The partial nucleotide sequences of 24 cDNA clones were determined and those of five clones containing poly(A) tail were compared with sequences of other plant viruses. One of these clones, V9, has a primary structure similar to the carlavirus group, suggesting that the clone V9 derived from a part of garlic latent virus (GLV). Northern blot analysis with the clone V9 as a probe demonstrated that GLV genome is 8.5 knt long and has a poly(A) tail. The clone V9 encodes coat protein (CP) of 33 kDa and nucleic acid binding protein of 10 kDa in different reading frame. The hexanucleotide motif, 5'-ACCUAA, which is conserved in the 3' noncoding region arid was proposed to be a cis-acting element involved in the production of negative strand genomic RNA was noticed. Complementary sequence to the hexanucleotide motif, 5'-TTAGGT, is also found in the positive strand of V9 RNA. The putative CP gene was cloned into the pRSET-A expression vector and expressed in E. coli BL21. The expressed recombinant V9CP protein was purified by $Ni^{2+}$ NTA affinity chromatography. The anti-V9CP antibody recognizes 34 kDa polypeptide which could be CP of GLV in infected garlic leaf extract. Immunoblot and Northern blot analysis of various cultivars shows wide occurrence of GLV in Korean garlic plants.

• Journal of Applied Biological Chemistry
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• v.54 no.2
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• pp.94-100
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• 2011

A gene encoding a putative phospholipase D was isolated from Bacillus licheniformis and cloned into pGEM-T easy vector. The gene was expressed in E. coli BL21 (DE3) using a pET-21(a) vector containing His6 tag. Affinity purification of the recombinant phospholipase D with nickel-nitrilotriacetic acid (Ni-NTA) resin resulted major one-band by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The purified enzyme showed a molecular weight of 44 kDa. The optimum activity of enzyme was around pH 7.0 and the enzyme was also the most stable around this condition. The optimum temperature was about $40-45^{\circ}C$ and the enzyme still showed considerable activities at wide range of temperature. Among various detergents, Triton X-100 significantly increased the enzyme activity, resulting in 181% activity of control at 0.6 mM of the detergent. Calcium ion did not significantly affect the enzyme activity, suggesting that the enzyme might be classified into $Ca^{2+}$-independent PLD.

• Research in Plant Disease
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• v.22 no.1
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• pp.32-37
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• 2016

In this work, major outer capsid protein (P10) encoded by genome segment S10 of Rice black-streaked dwarf virus (RBSDV) was expressed in Escherichia coli. Genomic dsRNA was extracted from RBSDV-miryang isolate infected rice plants. Based on the sequence of S10 (RBSDV-miryang, GenBank JX994211), a pair of S10 specific primers were designed and used to amplify the fragment encoding the N-part of P10. We amplified the partial gene (S10 1-834 nt) of RBSDV P10 (1-278 aa) by RT-PCR. Amplified RBSDV S10 (1-834 nt) was cloned into the expression vector pET32a (+). Recombinant RBSDV S10 (1-834 nt) was expressed in E. coli BL21(DE3) and purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity column. We successfully obtained P10 partial protein of RBSDV and the purified protein was used to immunize rabbits. The resulting polyclonal antiserum specifically recognized RBSDV from infected plant in both Western blotting and enzyme-linked immunosorbent assay. In this study, we provide purified RBSDV P10 (1-278 aa), which would be good material for the serological study of RBSDV-miryang isolates.