• Journal of Microbiology and Biotechnology
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• v.9 no.4
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• pp.386-392
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• 1999

A large-scale culture of hepatitis A virus in human diploid MRC-5 cells was conducted. In a roller bottle culture, the virus was grown to a maximum titer in 3 weeks after infection. Over 95％ of the cell-associated virus was excreted after culturing the infected cells in suspension media without fetal bovine serum for 3 days. The cultured virus was inactivated with formalin, concentrated by ultrafiltration, and partially purified by ultracentrifugation in a non-ionic gradient medium of Renocal. Two separate peak fractions showing high anti-HAY ELISA titer were pooled and about 40％ of HAV antigen was recovered by this purification procedure. Of the partially purified vaccine, the protein pattern in SDS-PAGE and immunogenicity in mice were compared with a commercial HAV vaccine. In SDS-PAGE, the purified vaccine in this study and the commercial vaccine showed almost the same protein pattern. The seroconversion rate of the purified vaccine in mice was not different from that of the commercial vaccine. Therefore, we could prepare a good grade of HAV vaccine by a simple purification procedure although the purification itself was not completed.

• Journal of Microbiology and Biotechnology
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• v.12 no.4
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• pp.635-642
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• 2002

To effectively investigate the identification and distribution of the lactic acid bacteria in Kimchi, polyphasic methods, including a PCR, SDS-PAGE of the whole-cell proteins, and 16S rRNA gene sequence analysis, were used. In various types of Kimchi fermented at 20$\^{C}$, the isolate KHU-31 was found to be the predominant lactic acid bacteria. This isolate was identified as Lactobacillus sake KHU-31, based on SDS-PAGE of the whole-cell proteins and a 165 rRNA gene sequence analysis, which provided accurate and specific results. Accordingly, the approach used in the current study demonstrated that Lactobacillus sake KHU-31, together with Leuconostoc mesenteroides, were the most predominant lactic acid bacteria in all types of Kimchi in the middle stage of fermentation at 20$\^{C}$.

• Biomolecules & Therapeutics
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• v.6 no.1
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• pp.89-94
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• 1998

CJ50001 is a recombinant human granulocyte colony-stimulating facto, (rHuG-CSF) that stimulates the formation of neutrophils from bone marrow stem cells. It was produced in E. colt and purified through refolding and several processes. We produced CS970125(300) using purified C150001 and additives in order to test the stability of CJ50001. When CS970125(300) was stored at 50'S for more than 1 week, high molecular weight proteins were formed and those proteins were detected by non-reducing SDS-PAGE, gel filtration HPLC, and Western blot. Those proteins showed single band at the same position of CJ50001 in reducing SDS-PAGE. These data indicated that those high molecular weight proteins were the multimers of C150001. In biological assays, iu viro and in viro, the multimers did not have biological activity and inhibitory action to that of CJ 50001. The mutimers did not induce toxicity in mice and rats in acute toxicity test. These results suggest that if Cs970125(300) containing CJ50001 is stored at 5$0^{\circ}C$, CJ50001 will be the multimers that do not have biological activity and inhibitory effect to CJ50001 and do not induce acute toxicity.

• Journal of Microbiology
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• v.42 no.3
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• pp.205-210
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• 2004

The sequence coding for carboxymethylcellulase (CMCase, CelC) was isolated from the DNA of Salmonella typhimurium URl. Comparison between the deduced amino acid sequence of CelC (368 amino acid residues, Molecular mass 41 kDa) and that of the previously published CMCase revealed that this enzyme belongs to the cellulase family 8 and D. The protein was overproduced in Escherichia coli using T7 expression system, and its activity was confirmed by CMC-SDS-PAGE. When the overexpressed CelC protein was tested on cellulose-type substrates, the recombinant protein is able to degrade cellulose-type substrates, such as CM-cellulose, xylan, avicel, lichenan, and laminarin. Optimal temperature and pH for enzyme activity were found to be 50$^{\circ}C$ and pH 6.5, respectively.

• Journal of fish pathology
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• v.6 no.2
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• pp.87-92
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• 1993

In February of 1990, an epizootic disease to masu salmon. Onchorynchus masou cultured at the hatchery of trout in Samchuk. Kwangwondo have broken out and induced heavy mortality. An infectious hematopoietic necrosis virus(IHNV) was isolated from diseased masu salmon fry by the use of fish cell line, CHSE-214. This IHNV isolated from masu salmon was compared with USA isolates of IHNV, SRCV and RB-76 by analysis of virion proteins in sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) and neutralization tests with two monoclonal antibodies raised against SRCV(MAb SRCV/A4) and RB-76(MAb RB/B5). In the antigenicity and the size of structural proteins. this IHNV, SCS atrain was smilar to RB-76 belonged to the electropherotype I proposed by Hsu et al.(1986).

• Korean Journal of Environmental Agriculture
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• v.28 no.4
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• pp.435-441
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• 2009

In the present study, we analyzed the defensin protein deduced from Korean radish (Raphanus sativus L.) seeds.To express the genes in E. coli, we constructed a recombinant expression vector with a defensin gene, named rKRs-AFP gene isolated from Korean radish seeds. Over expressed rKRs-AFP proteins was separated by SDS-PAGE to determine the purity, and protein concentration was determined by the Bradford method. Antifungal activity was assessed by disk assay method against the tested fungi. As a result, when 500 mL of cell culture were disrupted by sonicator, 32.5 mg total proteins were obtained. The purified protein showed a single band on SDS-PAGE with estimated molecular weight about 6 KDa, consistent with the molecular mass calculated from the deduced amino acid sequence. The purified rKRs-AFP protein showed remarkable antifungal activities against several fungi including Aspergillus niger, Botrytis cinerea causing the gray mold disease, and Candida albicans. In field tests using the purified rKRs-AFP protein, the protein showed the reducing activity of disease spot and the mitigating effect of spreading of disease like agrichemicals. The immuno-assay of rKRs-AFP protein showed that the purified protein entirely accumulated at B. cinerea cytoplasm through the hyphal septa shown by fluorescence imaging. There was no fluorescence inside the cell, when the hypha was incubated without the protein. These all results indicate that the recombinant rKRs-AFP proteins can be utilized as a potential antifungal drug to control harmful plant fungal pathogens.

• Nutrition Research and Practice
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• v.7 no.1
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• pp.3-8
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• 2013

Buckwheat is known as a health food but is one of the major food allergens triggering potentially fatal anaphylaxis in Asia, especially in Japan and Korea. This study was conducted to investigate the characteristic of enzymatic resistance of buckwheat protein and allergenic potential. Enzymatic resistance of buckwheat protein was performed with in vitro digestibility test in simulated gastric fluid (SGF), pH 1.2, using pepsin and simulated intestinal fluid (SIF) using chymotrypsin. Reactivity of buckwheat proteins to human IgE was performed using six allergic patients sensitized to buckwheat. Buckwheat's IgE levels were measured using the Phadia UniCAP-system. Buckwheat protein, 16 kDa, still remained after 30 min treatment of pepsin on SDS-PAGE. Even though 16 kDa almost disappeared after 60 min treatment, two out of the six buckwheat patients' sera showed reactivity to hydrolysate after 60 min treatment, indicating that allergenicity still remained. In simulated intestinal fluid (SIF) using chymotrypsin, buckwheat protein, 24 kDa, showed resistance to hydrolysis with chymotrypsin on SDS-PAGE, and still had allergenicity based on the result of ELISA. Our results suggest that buckwheat proteins have strong resistance to enzyme degradation. This may be attributed in part to the allergenic potential of buckwheat. Further study should be continued regarding buckwheat allergy.

• BMB Reports
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• v.39 no.1
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• pp.22-25
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• 2006

The gene encoding MPB83 from Mycobacterium bovis Vallee111 chromosomal DNA was amplified by using polymerase chain reaction (PCR) technique, and the PCR product was approximately 600bp DNA segment. Using T-A cloning technique, the PCR product was cloned into pGEM-T vector and the cloning plasmid pGEM-T-83 was constructed successfully. pGEM-T-83 and pET28a(+) were digested by BamHI and EcoRI double enzymes. The purified MPB83 gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-83 was constructed. Plasmid containing pET28a-83 was transformed into competence Escherichia coli BL21 (DE3). The bacterium was induced by isopropyl-$\beta$-D-thiogalactopyranoside (IPTG) and its lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 26 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed using Western-blotting. The results indicated that the protein was of antigenic activity of M. bovis. The results were expected to lay foundation for further studies on the subunit vaccine and DNA vaccine of MPB83 gene in their prevention against bovine tuberculosis.

• Applied Biological Chemistry
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• v.46 no.4
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• pp.385-390
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• 2003

An antimicrobial peptide was purified from the roots of Phytolacca americana L. and was designated as PAMP-r. Purification was carried out by DEAE-cellulose anion exchange, sephadex G-75 gel filtration, Mono S cation exchange, and Resource RPC reverse phage chromatography. The molecular weight of PAMP-r was estimated to be about 4,900 Da by 15% SDS-PAGE under reducing condition. PAMP-r exhibited a broad spectrum of antimicrobial activity. PAMP-r was stable against heat and pH treatment; its activity was not diminished by the heat treatment up to $80^{\circ}C$ for 30 min, and it showed a pH stability in the range between pH 3.0 to pH 8.0.

• Microbiology and Biotechnology Letters
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• v.29 no.4
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• pp.248-252
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• 2001

Recombinant Human serum albumin (rHSA) was purified to near homogeneity from H, polymorpha using heat treatment, ultrafiltratipn and Phenyl Sepharose CL-4B and Mono Q column chro - matographies with a recovery yield of 60% The molecular weight of the purified rHSA was estimated to be about 65,000 Da by denaturing SDS-PAGE The N terminal amino acid sequence of the purified HSA determined by Edman degradation was turned out to be Asp- Ala- His- Lys- Ser- Glu- Ala, suggesting that the rHSA expressed in H, polymorpha was efficiently secreted and correctly processed at the cleavage site of secretion signal sequence. The purified human albumin showed the pI value identical to that of authentic human serum albumin.