• Title/Summary/Keyword: 1-D SDS PAGE

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Fibrinolytic Activity and Proteomic Analysis of Bacillus licheniformis HK-12 Isolated from Chungkuk-Jang (청국장에서 분리한 Bacillus licheniformis HK-12의 혈전용해활성과 프로테옴 분석)

  • Sohn, Byung-Hee;Kwon, Sang-Chul;Oh, Kye-Heon
    • Journal of the Korea Academia-Industrial cooperation Society
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    • v.9 no.3
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    • pp.800-806
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    • 2008
  • The strain HK-12 was enriched and isolated from naturally fermented soybean for the production of fibrinolytic enzyme and the proteome of this enzyme induced during the incubation period was analyzed. The activity of fibrinolytic enzyme derived from supernatants of the HK-12 culture was performed by fibrin plate method for solid fibrinolytic activity. As the result, the fibrinolytic activity of HK-12 grown on the nutrient agar media was about 2.3 times greater than that of plasmin used as standard. The purified enzyme was prepared by a series of purification process including ammonium sulfate precipitation, DEAE-cellulose, Sephadex chromatography. The molecular weight of the enzyme was determined to approximately 23kDa with SDS-PAGE. In order to examine which strain HK-12 proteins increased or decreased during the incubation period, 2-DE analysis was performed. Protein spot #1 significantly expressed on the 2-DE gel of bacteria cultivated for 36-hrs was analysed. As the result of protein sequence analysis using MALDI-TOF MS, one protein was identified as serine protein kinase (PrkA).

Biochemical and Genetic Variation of Hordein Subunits in Korean Barley

  • Lee, Sung-Shin;Kim, Jin-Baek;Kim, Dong-Sub;Nam, Jung-Hyun;Kim, Jae-Chul;Kim, Chung-Kon;Hong, Byung-Hee;Seo, Yong-Weon
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.46 no.2
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    • pp.100-105
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    • 2001
  • One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D SDS-PAGE) was used to determine whether it would provide improved resolving power of hordein proteins concomitant with improved identification of Korean barley cultivars and germplams. This system gave rapid and reproducible separations of hordein polypeptides. Total fourteen of clear and easily scorable subunits were identified in Korean barley cultivars and germplasms and their polymorphic constitutions could provide biochemical genetic information in progeny analysis and endosperm quality improvement in barley breeding programs. Each hordein polypeptides residing in B, C, and D hordein pattern designations were scored to prepare a cultivar catalogue of protein patterns. On the basis of this character, 7 hordein polypeptide patterns were constructed from 108 barley cultivars and experimental lines. The molecular weight of hordein subunits in Korean barley cultivars and experimental lines varied in the range of 98 to 48 kDa. In contrast, less polymorphic hordein polypeptides were found in the low protein barley lines including malting barleys than those found in Korean barley cultivars and experimental lines.

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Disassembly of Chlorophyll-Protein Complexes in Arabidopsis thaliana during Dark-Induced Foliar Senescence

  • Lee, Choon-Hwan
    • Journal of Plant Biology
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    • v.39 no.4
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    • pp.301-307
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    • 1996
  • The disassembly of Chl-protein complexes during dark-induced senescence (DIS) was investigated using detached third and fourthleaves of 21$\pm$1 day-old Arabidopsis thaliana. Although Chl content decreased linearly after 1 d, a significant decrease of photochemical effeciency (Fv/Fm) was observed after 2 d. In experiments using native green gel electrophoresis of Chl-protein complexes combined with additional two-dimensional SDS-PAGE analysis, we could observe the degradation of both photosystems after 2 d. Although light-harvesting complex(LHC) for PSI (LHCI) was degraded first in PSI complex, small PSII apoproteins including CP47/CP43 and D1/D2 apoproteins were degraded first in PSII complexes. LHC for PSII (LHCII) trimers were stable until 4 d. The level of LHCII monomers was increased until 3 and decreased thereafter, resulting in the increase of free pigments. These results suggest that the disassembly process of PSI is different from that of PSII.

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Elctrophoretic Properties of Heat-Induced Bovine α-Lactalbumin

  • Lee, You-Ra;Hong, Youn-Ho
    • Preventive Nutrition and Food Science
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    • v.7 no.1
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    • pp.48-51
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    • 2002
  • In order to study the reaction behavior of bovine holo- and apo-$\alpha$-lactalbumin ($\alpha$-La) during heat treatment at 65~10$0^{\circ}C$, the samples were analysed by first (ID)-and second-dimensional (2D) native-polyacrylamide gel electrophoresis (Native-PAGE) and sodium dodecylsulfate (SDS)-PAGE. When bolo-$\alpha$-La or apo- $\alpha$ -La were heated, they formed non-native, monomers, dimers and trimers. The apo-$\alpha$-La was more heat-sensitive than holo-$\alpha$-La. The monomers seemed to have the same composition as the native $\alpha$-La, but many of the disulfide bonds could be non-native.

Changes of Lectin from Viscum coloratum by Fermentation with Lactobacillus plantarum -Isolation and Purification- (유산균 발효에 의한 겨우사리 중의 렉틴 성분의 변화 -분리 및 정제-)

  • Park, Won-Bong;Kim, Hee-Sook
    • YAKHAK HOEJI
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    • v.38 no.6
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    • pp.687-695
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    • 1994
  • Lectin from mistletoe(Viscum coloratum) fermented by Lactobacillus plantarum for 1,2,3 days were obtained by salt fractionation, gel filtration, anion exchange chromatography and SDS-PAGE, and compared with the lectin from unfermented mistletoe. The new lectin of molecular weight of about 18,500D from fermented mistletoe was identified.

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Application of Gel-based Proteome Analysis Techniques to Studying Post-mortem Proteolysis in Meat

  • Hwang, I.H.
    • Asian-Australasian Journal of Animal Sciences
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    • v.17 no.9
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    • pp.1296-1302
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    • 2004
  • This study was conducted to evaluate the possible application of 2 D-SDS-PAGE (2 DE)-based proteome analysis techniques to the assessment of extreme proteolysis in postmortem skeletal muscle. Eight Hanwoo longissimus muscles were incubated immediately after slaughter for 24 h at 5$^{\circ}C$, 15$^{\circ}C$ or 36$^{\circ}C$. Warner Bratzler (WB)-shear force and ultrastructural configuration were determined at 24 h, and rate of proteolysis to 24 h was determined by 1 D-SDS-PAGE (1 DE) and 2 DE. In addition, tentative protein identification was performed from peptide mass fingerprints of MALDI-ToF analysis of major protein groups on 2 DE profiles. The result showed that although ultrastructural configuration was similar between the 5$^{\circ}C$ and 36$^{\circ}C$ treatments, meat at 5$^{\circ}C$ had higher WBshear force (approximately 5 kg greater). A higher rate of protein degradation at 36$^{\circ}C$ was observed based on Troponin-T degradation, 1 DE, and 2 DE analysis. This indicates that proteolysis during the early postmortem period was a significant determinant of shear force at 24 h. Little difference in proteolysis between 5$^{\circ}C$ and 15$^{\circ}C$ treatments was found based on classic 1 DE profile assessment. Meanwhile, considerable differences in the 2 DE profiles between the two treatments were revealed, with substantially higher rate of proteolysis at 15$^{\circ}C$ compared to 5$^{\circ}C$. Nuclease treatment improved 2 DE profile resolution. 400 ${\mu}$g and 600 ${\mu}$g of sample loading appeared to be appropriate for 24 cm pH 3-10 and pH 5-7 IPG strips, respectively. Protein detection and quantification of the 5$^{\circ}C$, 15$^{\circ}C$ and 36$^{\circ}C$ 2 DE profiles revealed 78, 163 and 232 protein spots respectively that were differentially modified in terms of their electrophoretic properties between approximately pI 5.3-7.7 with the molecular weight range of approximately 71-12 kDa. The current results demonstrated that 2 DE was a superior tool to 1 DE for characterising proteolysis in postmortem skeletal muscle.

Purification and Characterization of $\beta$-Xylosidase B of Bacillus stearothemophilus No.236 Produced by Recombinant Escherichia coli. (재조합 균주 Escherichia coli가 생산하는 Bacillus stearothermophilus No.236 $\beta$-Xylosidase B의 정제 및 특성)

  • 장욱진;조쌍구;최용진
    • Microbiology and Biotechnology Letters
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    • v.26 no.4
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    • pp.297-302
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    • 1998
  • $\beta$-Xylosidase B was produced by Escherichia coli HB101/pKMG12 carrying the xylB gene of Bacillus stearothermophilus No.236 on its recombinant plasmid. The $\beta$-xylosidase B produced was purified by ammonium sulfate fractionation, DEAE-Sepharose CL-6B, Sephacryl S-200 and Superdex 200 HR gel filtration. The purified enzyme showed the highest activity at pH 6.5 and 5$0^{\circ}C$. But, the enzyme was observed to be very sensitive to the pH and temperature of the reaction mixture. The enzyme was activated about 35% of its original activity in the presence of 1 mM of $Mn^{2+}$ but it was completely inhibited by $Ag^{+}$, $Cu^{2+}$and $Hg^{2+}$ions. In contrast with the $\beta$-xylosidase A, the B enzyme was found to have $\alpha$-arabinofuranosidase activity though the activity was fairly low compared with the $\alpha$-arabinofuranosidase produced from the arfI gene of the same Bacillus stearothermophilus. Therefore, $\beta$-xylosidase B is considered to be more suitable than $\beta$-xylosidase A at least for the biodegradation of arabinoxylan. The $K_{m}$ and V$_{max}$ values of the $\beta$-xylosidase B for o-nitrophenyl-$\alpha$-D-xylopyranoside were 6.43 mM and 1.45 $\mu$mole/min, respectively. Molecular mass of the enzyme was determind to be about 54 kDa by SDS-PAGE and 160 kDa by Superdex 200HR gel filtration, indicating that the functional $\beta$-xylosidase B was composed of three identical subunits.s.

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Cellular Responses and Morphological Changes of RDX-degrading Bacterium, Pseudomonas sp. HK-6 Exposed by Explosive Hexahydro-1,3,5-triaitro-1,3,5-triazine (RDX). (폭약 Hexahydro-1,3,5-trinitro-1,3,5-triazine(RDX)에 노출된 분해세균 Pseudomonas sp. HK-6의 세포반응과 형태변화)

  • 장효원;강형일;김치경;오계헌
    • Microbiology and Biotechnology Letters
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    • v.31 no.1
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    • pp.75-82
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    • 2003
  • The cellular responses of RDX-degrading bacterium, Pseudomonas sp. HK-6 to explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) were examined. Strain HK-6 grown at different RDX concentrations was found to demonstrate the survival rate in proportional to the rate of the stress shock proteins produced in this bacterium. Analysis of total cellular fatty acid acids showed that lipids 10:0 iso and 14:1 $\omega$5c/$\omega$5t increased approx three times in strain HK-6 grown on RDX media than TSA media. SDS-PAGE and Western blot using anti-DnaK and GroEL revealed that several stress shock proteins including 70 kDa DnaK and 60 kDa CroEL were newly synthesized in strain HK-6 exposed to different RDX concentrations in exponentially growing cultures. 2-D PAGE of soluble protein fractions from the culture of HK-6 exposed to RDX demonstrated that approximately 300 spots were observed on the silver stained gel ranging from pH 3 to pH 10. As a result, 10 spots were significantly induced and expressed in response to RDX. Scanning electron microscopy fur the cells treated with 0.135 mM RDX for 12 hrs showed the presence of perforations and irregular rod shapes with wrinkled surfaces.

Cloning and Characterization of Pseudomonas mucidolens Exoinulinase

  • Kwon, Young-Man;Kim, Hwa-Young;Choi, Yong-Jin
    • Journal of Microbiology and Biotechnology
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    • v.10 no.2
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    • pp.238-243
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    • 2000
  • An exoinulinase (${\beta}-D-fructofuranosidase$) gene was cloned by chromosome walking along the upstream region of the endoinulinase gene of Pseudomonas mucidolens isolated from soil. the exoinulinase gene consisted of an ORF of 0,506 bp encoding a polypeptide of 501 amino acids with a deduced molecular weight of 55,000. The exoinulinase produced by the recombinant Escherichia coli $DH5{\alpha}$ strain was also purified to homogeneity as determined by SDS-PAGE and a zymogram. The molecular weight of the purified exoinulinase according to both SDS-PAGE and gel filtration matched the deduced molecular weight of the protein described above, thereby indicating that the native form of the exoinulinase was a monomer. The purified enzyme hydrolyzed activity value of 2.0. Furthermore, no inulo-oligomers were liberated from the inulin substrate in the enzymatic reaction mixtures incubated for 90 min at $55^{\circ}C$. Taken together, these results indicate that the purified ${\beta}-D-fructofuranosidase$ was an exoinulinase. The pH and temperature optima of the exoinulinase were pH 6.0 and $55^{\circ}C$, respectively. the enzymehad no apparent requirement for a cofactor, and its activity was completely inactivated by $Ag^{+},{\;}Hg^{2+},{\;}and{\;}Zn^{2+}$. Kinetic experiments gave $K_m,{\;}V_{max},{\;}and{\;}K_{cat}$ values for inulin of 11.5 mM, 18 nM/s, and $72{\;}s^{-1}$, respectively. the exoinulinase was fairly stable in broad pH conditions (pH 5-9), and at pH 6.0 it showed a residual activity of about 70% after 4 h incubation at $55^{\circ}C$.

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Production of a Recombinant Anti-Human CD4 Single-Chain Variable-Fragment Antibody Using Phage Display Technology and Its Expression in Escherichia coli

  • Babaei, Arash;Zarkesh-Esfahani, Sayyed Hamid;Gharagozloo, Marjan
    • Journal of Microbiology and Biotechnology
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    • v.21 no.5
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    • pp.529-535
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    • 2011
  • Single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulin, connected with a short linker peptide of 10 to about 20 amino acids. In this study, the scFv of a monoclonal antibody against the third domain of human CD4 was cloned from OKT4 hybridoma cells using the phage display technique and produced in E. coli. The expression, production, and purification of anti-CD4 scFv were tested using SDS-PAGE and Western blot, and the specificity of anti-CD4 scFv was examined using ELISA. A 31 kDa recombinant anti-CD4 scFv was expressed and produced in bacteria, which was confirmed by SDS-PAGE and Western blot assays. Sequence analysis proved the ScFv structure of the construct. It was able to bind to CD4 in quality ELISA assay. The canonical structure of anti-CD4 scFv antibody was obtained using the SWISS_MODEL bioinformatics tool for comparing with the scFv general structure. To the best of our knowledge, this is the first report for generating scFv against human CD4 antigen. Engineered anti-CD4 scFv could be used in immunological studies, including fluorochrome conjugation, bispecific antibody production, bifunctional protein synthesis, and other genetic engineering manipulations. Since the binding site of our product is domain 3 (D3) of the CD4 molecule and different from the CD4 immunological main domain, including D1 and D2, further studies are needed to evaluate the anti-CD4 scFv potential for diagnostic and therapeutic applications.