• Title/Summary/Keyword: 1-D SDS PAGE

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Iron Uptake by the Recombinant Yeasts Producing Ferritin Heteropolymers (재조합 효모에 의한 Ferritin 이형집합체의 발현과 철 흡수)

  • Chang Yu Jung;Park Chung Ung;Kim Kyung Suk
    • KSBB Journal
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    • v.19 no.5
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    • pp.352-357
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    • 2004
  • Human ferritin H- and L-chain genes (hfH and hfL) were cloned into the yeast shuttle vector YEp352 containing the GAL1 (galactokinase) and GAL10 (epimerase) divergent promoters and the vectors constructed were used to transform Saccharomyces cerevisiae 2805. SDS-PAGE displayed expression of the introduced hfH and hfL in both recombinant strains of Y1H10L and Y1L10H. The ferritin subunits, that represented ca. $22\%$ and $15\%$ of the soluble proteins in Y1H10L and Y1L10H, were spontaneously assembled into active ferritin heteropolymers. The H subunit content of the purified recombinant human ferritin heteropolymers was proven to reflect the relative expression yield of the subunits. When the cells of 2d culture were incubated with 14.3 mM Fe(2), the cellular iron concentration of Y1H10L and Y1L10H was 1.7 and 2.0 times, respectively, that of the control strain. It is assumed that increase in the iron uptake of the recombinant yeasts is closely related to ferritin expression and H subunit content.

Comparison of Characteristics of Hepatic Microsomal Cytochrome P45O-dependent Monooxygenases from Snake and Rat (꽃뱀과 흰쥐의 간 마이크로좀에 존재하는 Cytochrome P45O 의존성 Monooxygenases의 특성 비교)

  • Ja Young Moon;Dong Wook Lee;Ki Hyun Park
    • Journal of Life Science
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    • v.8 no.6
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    • pp.695-701
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    • 1998
  • This study was carried out to investigate levels of the components of microsomal mixed function oxidase (MFO) system and activities of the hepatic microsomal cytochrome P45O (P45O)-dependent monooxygenases of grass snake (Natrix tigrina Lateralis) and to compare with those of rat. The levels of P45O and cytochrome b$_{5}$, (b$_{5}$) of snake were much lower than those in rat. NADPH-cytochrome c reductase activity in the snake was also only 40% of that in the rat. Activities of 7-ethoxycoumarin 0-deethylase (ECOD) and benzphetamine N-demethylase (BPDM) of snake hepatic microsomes, when compared with those of rat, were markedly low. But, aryl hydrocarbon hydroxylase (AHH) and testosterone hydroxylase (TSH) activities were nearly the same or higher than those of the rat. Of the P45O-dependent TSHs measured, 7$\alpha$-hydroxylase activity was the highest in snake, whereas, 6$\beta$-hydroxylase activity was the highest in rat. However, stereoselectivity of the enzyme from the snake to C2 and C6 positions of testoste-rone was the same as rat. The result of radioimmunoassay (RIA) for the identification of five P45O isozymes with MAbs shows that relatively high content of ethanol-inducible P45O isozyme, CYP2El, exists in the rat, whereas MC-inducible P45O isozyme, CYP2A1/1A2, does in the snake. From the analyses of SDS-PAGE and RIA of partially pu-rified P45O, we suggest the possibility of the presence of a certain P45O isozyme(s) in hepatic microsomes of snake different from those of rat.

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An Experimental Study on Production of Egg Yolk Antibody(IgY) against Bee Venom (봉독의 항독소(IgY)생산을 위한 실험적 연구)

  • Hwang, Tae-Jun;Lee, Seung-Bae;Gwon, Gi-Rok
    • Journal of Pharmacopuncture
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    • v.4 no.2
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    • pp.5-15
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    • 2001
  • This study was carried out for production of neutral antibody to bee venom $(anti-phospholipase\;A_2IgY)$. Hen layings were injected repeatedly with bee venom and phospholipase $A_2$ with Freund's adjuvant. Specific antibody in egg yolk from immunized hen laying was separated, and purified, also immunological characteristics of anti phospholipase $A_2\;IgY$ was invested. The results were summarized as follows: 1. Phospholipase $A_2$ was showed single band at molecular weight 17,000 in SDS-PAGE and bee venom was showed two band at molecular weight 17,000 and under molecular weight 6,500 in SDS-PAGE. 2. During 70 days after hen immunized with bee venom and phospholipase $A_2$, antibodies(anti-bee venom IgY) to bee venom were showed poor ELISA value in egg yolk, but antibodies$(anti-Phospholipase\;A_2IgY)$ to phospholipase $A_2$ in egg yolk were increased ELISA value from 8 days or 15 days and found maximum ELISA value at 42 days. Also after booster at 49 days, ELISA value of anti Phospholipase $A_2\;IgY$ in egg yolk was supported at optical density(O.D) 1.0 level, continuously. 3. Titer of phospholipase $A_2\;IgY$ was showed 1: 32,000. 4. In double immunodiffusion test to phospholipase $A_2$ after double dilution of anti-phospholipase $A_2\;IgY$, only precipitation line was made in 1:1 dilution well of anti-Phospholipase $A_2\;IgY$. But In immunodiffusion test to anti-phospholipase $A_2\;IgY$ after double dilution of phospholipase $A_2$, Precipitation line to 250ul/ml well of phospholipase $A_2$ was showed. In double immunodiffusion test to bee venom(1mg/ml) after double dilution anti-phospholipase $A_2\;IgY$, all well without 1:32 dilution well were showed strong precipitation line. 5. In dot bloting test to anti-phospholipase $A_2\;IgY$ after diluting bee venom(0.5mg/ml), dot bloting color was showed clearly to $1/100(5{\mu}g/ml)$ in bee venom.

Comparison of Meat Quality Characteristics of Wet- and Dry-aging Pork Belly and Shoulder Blade

  • Hwang, Young-Hwa;Sabikun, Nahar;Ismail, Ishamri;Joo, Seon-Tea
    • Food Science of Animal Resources
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    • v.38 no.5
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    • pp.950-958
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    • 2018
  • The physicochemical characteristics and oxidative stability of wet-aged and dry-aged pork cuts were investigated at different aging periods (1, 7, 14 and 21 d). Samples were assigned into four groups in terms of shoulder blade-wet aging (SW), shoulder blade-dry aging (SD), belly-wet aging (BW), and belly-dry aging (BD). SD showed significantly higher pH at 21 d of aging than the other samples. Wet-aged cuts had significantly higher released water (RW) %, lightness ($L^*$) and shear force compared to the dry-aged meats. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed greater degradation of proteins for dry-aged cuts than the wet-aged cuts. At the end of aging, wet-aged cuts showed significantly lower thiobarbituric acid-reactive substances (TBARS) value than the dry-aged samples, indicating higher oxidative stability for wet-aged pork cuts. However, dry-aging led to higher degradation of proteins resulting in increased water-holding capacity (WHC) and decreased shear force value.

The Functional Role of Maturation Promoting Factor in the Two-cell Embryos (생쥐 2-세포기 배아에서 성숙유도물질의 기능적 역할)

  • 강해묵;이대기
    • The Korean Journal of Zoology
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    • v.36 no.2
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    • pp.277-284
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    • 1993
  • A possible involvement of maturation promoting factor (nfPF) in the two-cell block phenomenon was studied by fusion experiments. Germinal vesicle (GlF) ooeyte was fused with a blastomore from late or blocked 2-cell mouse embryos. and germinal vesicle breakdoum (GVBD) of fused GV oocvtes in the presence of dbcAMP (100$\mu$g/ml) was scored as an index of MPF aniviD. GnD was induced approximately 30% by fusion of a blastomere derived from late 2-cell embryos, but not from blocked 2-cell embryos. The rate of GVBD was changed when GV oocyte was fused with a blastomere from late 2-cell embryos which were treated with u-amanitin, puromvcin or colcemid before and after hsion: Treatment of late 2-cell embryos with puromycin (50 Is/mll but not with u-amanitin (100 Is/ml) clearly inhibited GVBD, indicating that do novo protein synthesis maw be required for the appearance of MPF activity in late 2-cell embryos. Treatment of late 2-cell embryos w기h colcemid (0.1 Is/mll doubled GVBD, presumably due to the maintenance of metaphase or mitotic phase. SDS-PAGE and twoiimensional electrophoresis revealed that there was no difference in protein synthetic pattern in late and blocked 2-cell embryos, but three phosphoproteins with 27, 35 and 46 M)a, presumsblv M-phase components were phosphorylated in late 2-cell embryos but not in blocked 2-cell embryos. It seems then that MPF activity is closely related to phosphorylstion of M-phase components in late 2-cell embryos.

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Cloning of a Bacillus subtilis WL-7 Mannanase Gene and Characterization of the Gene Product

  • KWEUN , MIN-A;LEE, MI-SUNG;CHOI, JOON-HO;CHO, KI-HAENG;YOON, KI-HONG
    • Journal of Microbiology and Biotechnology
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    • v.14 no.6
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    • pp.1295-1302
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    • 2004
  • A gene encoding the mannanase of Bacillus subtilis WL-7, which had been isolated from Korean soybean paste, was cloned into Escherichia coli, and the gene product was purified from the culture filtrate of the recombinant E. coli. This mannanase gene, designated manA, consisted of 1,086 nucleotides, encoding a polypeptide of 362 amino acid residues. The deduced amino acid sequence was highly homologous to those of mannanases belonging to the glycosyl hydrolase family 26. The molecular mass of the purified mannanase was 38 kDa as estimated by SDS-PAGE. The enzyme had a pH optimum at 6.0 and a temperature optimum at $55^{\circ}C$. The enzyme was active on locust bean gum, konjak, guar gum, and lichenan, while it did not exhibit activity towards yeast mannan, laminarin, carboxymethylcellulose, $\beta$­glucan, xylan, and para-nitrophenyl-$\beta$-mannopyranoside.

Proteomic Assessment of Dung Beetle, Copris tripartitus Immune Response

  • Suh, Hwa-Jin;Bang, Hea-Son;Kim, Seong-Ryul;Yun, Eun-Young;Park, Kwan-Ho;Kang, Bo-Ram;Kim, Ik-Soo;Jeon, Jae-Pil;Hwang, Jae-Sam
    • International Journal of Industrial Entomology and Biomaterials
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    • v.17 no.2
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    • pp.217-221
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    • 2008
  • Dung beetle larvae at the $3^{rd}$ instar were injected with lipopolysaccaride and inducible proteins were examined within a pI level of 3-10 and a size level by proteomics, including 1-D SDS PAGE analysis and antibacterial assay. The immune infected larvae extracts provided seven protein bands in one-dimensional electrophoresis and its antibacterial activity also checked. Hemolymph protein from immune infected larvae of the dung beetle were separated by twodimensional gel electrophoresis and compared with those from native larvae. In 2-D gel electrophoresis, we detected 63 immune infected unique and 32 up-regulated proteins, and 36 proteins that were down-regulated or not present in treated gel. Ten protein spots from unique proteins and those presented as different level of abundance in infected and native larvae were specially expressed. These differentially expressed proteins were proposed to be involved in the defense mechanism against microorganism.

Purification and Characterization of a New Galactoside Specific Lectin from Trichosanthes kirilowii Root

  • Yun, Doo-Hee;Park, Eun-Ju;Park, Jong-Ok;Lee, Young-Han;Seo, Jeong-Kon;Ryu, Sung-Ho;Suh, Pann-Ghill;Kim, Hee-Sook
    • BMB Reports
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    • v.28 no.1
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    • pp.6-11
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    • 1995
  • A new lectin, named TRA, was purified from Trichosanthes kirilowii root by acid-treated Sepharose 6B, Mono-Q, and TSK-gel 3000SW column sequential chromatography. The lectin appeared homogeneous by native gel electrophoresis at pH 4.3 and gave two protein bands of Mr=31 and 28 kDa by SDS-PAGE. The N-terminal amino acid sequences of the polypeptides of TRA have not been reported in amino acid sequences of the lectins. TRA lectin formed a precipitate with asialofetuin, neuraminidase-treated fetuin. A sugar inhibition assay indicated that N-acetyl-D-galactosamine, among the monosaccharides tested, was the most potent inhibitor of TRA-induced hemagglutination. Asialofetuin showed a 260-times stronger inhibitory activity than N-acetyl-D-galactosamine. TRA lectin also showed agglutination with normal leukocytes and lymphoma cells, but not with premature hemopoietic cells. These results suggest that TRA is a novel plant lectin.

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A Comparison of Protein Characteristics of Korean and Imported Wheat Varieties (한국산 소맥과 수입 소맥의 단백질 특성 비교)

  • Koh, Bong-Kyung
    • Korean Journal of Food Science and Technology
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    • v.31 no.3
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    • pp.586-592
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    • 1999
  • The objective of this study was to investigate the fundamental characteristics of proteins effecting on Korean wheat flour quality. Mixing properties and protein contents of Eunpa, Allgreu, and Woori showed soft wheat characteristics. Tapdong had strong dough stability and high protein content like hard wheat flour. However, even though Greu had a high protein content, the dough strength was very weak like soft wheat flour. No major difference was observed in protein solubility with water, 0.1N NaCl, 70% ethyl alcohol, 0.1 N acetic acid solution and in sulfur and disulfide contents between Korean wheat flours and imported flours. However, DNS (Dark Norther Spring) had more contents of acid insoluble and SDS insoluble proteins compared with Korean wheat varieties. PAGE pattern of Tapdong was very similar to imported wheat flours at the high molecular weight regions and characteristic high molecular weight glutenin subunits were observed over 116 kD protein molecular weight region from 4 Korean wheat flours such as Eunpa, Woori, Allgreu, and Greu.

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Purification of Alginate Lyase from Streptomyces violaceoruber and the Growth Activity of Intestinal Bacteria by Degree of Polymerization of Alginate Hydrolysates (Streptomyces violaceoruber 유래 Alginate Lyase의 정제 및 Sodium Alginate 가수분해 올리고당의 중합도별 Bifidobacterium spp.과 Lactobacillus spp.에 대한 생육활성)

  • Yoon, Min;Park, Young-Seo;Park, Gwi-Gun
    • Food Engineering Progress
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    • v.21 no.2
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    • pp.103-109
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    • 2017
  • Alginate lyase from Streptomyces violaceoruber was purified by DEAE sephacel chromatography and SP sepharose chromatography. The specific activity of the purified enzyme was 14.6 units/mg protein, representing a 40.6-fold purification of the crude extract. The final preparation thus obtained showed a single band on Tricine-SDS polyacrylamide gel electrophoresis whose molecular weight was determined to be 23.3 kDa. The polyMG block of sodium alginate was hydrolyzed by the purified alginate lyase and then separated by activated carbon column chromatography and bio gel P-2 gel filtration. The main hydrolysates were composed of hetero type M/G-oligosaccharides with the degrees of polymerization (D.P.) being 6 and 8. To investigate the effects of hetero type M/G-oligosaccharides from the sodium alginate on the growth of some intestinal bacteria, cells were cultivated individually on the modified-MRS medium containing D.P. 6 and 8 M/G-oligosaccharides. B. longumgrew 4.25-fold and 6.44-fold more effectively by the treatment of D.P. 6 and 8 M/G-oligosaccharides compared with those of standard MRS medium. In addition, B. bifidumgrew 3.3-fold and 5.4-fold more effectively by the treatment of D.P. 6 and 8 M/G-oligosaccharides. In conclusion, D.P. 8 was more effective than D.P. 6 hetero M/G-oligosaccharides as regards the growth of Bifidobacteriumspp. and Lactobacillus spp.