• 제목/요약/키워드: E6/E7 protein

검색결과 816건 처리시간 0.033초

무지개송어의 간세포 초대배양에 의한 Vitellogeinin 합성 유도 (Vitellogenin Induction by Rainbow trout (Oncorhynchus mykiss) Hepatocytes in Primary Culture)

  • 여인규
    • 한국양식학회지
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    • 제11권4호
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    • pp.557-564
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    • 1998
  • 무지개송어의 초대배양 간세포에 있어서 Vitellogenin (VTG)의 합성 유도는 전기영동적 수법에 의해 행하였다. 간세포는 7일 동안 phositively charged dish를 이용한 배양으로 단층 확산되었다. 배양 7일의 간세포 생존율은 $E_2$의 유무에 따라 각각 20.7% 및 23.6%가 감소하였으며, DNA함유량도 각각 13.7% 및 14.0%가 감소하였다. $E_2$ 첨가군과 대조군간에는 생존율과 DNA 함유량에 있어서는 유의한 차이가 나타나지 않았다. 그리고, 총 단백질에 대한 VTG의 비율은 $E_2$$10^{-6}$ M의 첨가시에 최대치를 나타내었다. 그러나, 고농도($10^{-5}$M)에서는 오히려 감소하는 경향을 나타내었다.

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효소 저해법을 이용한 유기인계 및 Carbamate계 농약의 다성분 잔류 검출 (Detection for Multiresidue of the Organophosphorus and Carbamate Pesticides by Enzyme-Inhibition Method)

  • 김정호
    • Environmental Analysis Health and Toxicology
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    • 제17권3호
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    • pp.265-272
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    • 2002
  • Enzyme-Inhibition방법으로 다성분 잔류 농약의 검출 기법을 개발하기 위해, 음용수 허용기준 설정 농약인 유기인계 농약으로 malathion, parathion, diazinon과 carbamate농약으로 carbary에 대한 acetylcholinesterase (AChE)과 cholinesterase (ChE) 활성저해 관계를 규명하였다. 병아리 뇌의 AChE와 ChE 활성도는 각각 166.6 및 5.8$\mu$mol/min/g protein이었고, 혈장에서는 각각 23.1$\mu$mol/min/g protein과 8.3 $\mu$mol/min/g protein 이었다. AChE와 ChE의 최적 PH는 각각 8.2및 7.8 이었다. Km은 0.034 및 0.045 mM 이었다. 유기인계농약에서 AChE와 ChE의 I$_{50}$ 값의 malathion이 55.82 및 99.42mg/L이었고, Parathion은 31.16및 29.13mg/L이었고, diazinon은 17.89 및 19.62 mg/L 이었다. Carbamate농약인 carbaryl의 AChE와 ChE의 I$_{50}$ 값의 0.10및 0.05mg/L이었다. 먹는 물 관리법에 의한 carbaryl의 먹는 물 허용 수질기준인 0.07mg /L을 AChE와 ChE의 I$_{50}$에서 검출할 수 있다. AChE및 ChE을 이용한 enzyme-inhibition(EI)법은 carbamate농약인 carbaryl을 먹는 물 허용 수질기준인 0.07mg/L가지 검출 할 수 있으므로, 다성분 잔류분석법 (MRM, Multiresidue Method)으로 이용할 수 있다. 따라서 Enzyme lnhibition방법을 이용하여 자연환경 중 carbamate계 농약을 쉽고 빠르게 검출할 수 있는 새로운 bioassay법으로 응용할 수 있다. 수 있다.

Expression of orf7(oxi III) as dTDP-Glucose 4,6-Dehydratase Gene Cloned from Streptomyces antibioticus Tu99 and Biochemical Characteristics of Expressed Protein

  • Yoo, Jin-Cheol;Han, Ji-Man;Sohng, Jae-Kyung
    • Journal of Microbiology and Biotechnology
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    • 제9권2호
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    • pp.206-212
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    • 1999
  • The gene orf7(oxi III) was expressed using an E. coli system in anticipation that it would encode dTDP-glucose 4,6-dehydratase which is involved in the biosynthesis of the olivose moiety of chlorothricin produced from Streptomyces antibioticus Tu99. The solubility of the expressed protein increased up to 20% under optimal induction conditions. The expressed protein was purified from the E. coli BL 21(DE3) cell lysate by a 28.5-fold purification in two chromatography steps with a 38% recovery to near homogeneity. The molecular weight and N-terminal amino acid sequence of the purified protein correlated with the predicted mass and sequence deduced from the orf7 gene. The purified protein was a homodimer with a subunit relative molecular weight of 38,000 Dalton. The expressed protein was found to exhibit dTDP-glucose 4,6-dehydratase activity and be highly specific for dTDP-glucose as a substrate. The values of K'm and V'max for dTDP-glucose were 28 $\mu$M and 295 nmol $min^{-1} (mg protein)^{-1}$, respectively. dTTP and dTDP were strong inhibitors of this enzyme.$NAD^+$, the coenzyme for dTDP-glucose 4,6-dehydratase, was tightly bound to the expressed protein.

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Protein Evaluation of Dry Roasted Whole Faba Bean (Vicia faba) and Lupin Seeds (Lupinus albus) by the New Dutch Protein Evaluation System: the DVE/OEB System

  • Yu, P.;Egan, A.R.;Leury, B.J.
    • Asian-Australasian Journal of Animal Sciences
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    • 제12권6호
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    • pp.871-880
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    • 1999
  • The effects of dry roasting (110, 130, $150^{\circ}C$ for 15, 30, 45 min) on potential ruminant protein nutritional values in terms of: a), rumen bypass protein (BCP); b), rumen bypass starch (BST); c), fermented organic matter (FOM); d), true absorbed bypass protein (ABCP); e) microbial protein synthesized in the rumen based on available energy (E_MP); f), microbial protein synthesized in the rumen based on available nitrogen (N_MP); g), true protein supplied to the small intestine (TPSI); h), true absorbed rumen synthesized microbial protein (AMP); i), endogenous protein losses (ENDP); j), true digested protein in the small intestine (DVE); k), degraded protein balance (OEB) of whole lupin seeds (WLS) and faba beans (WFB) were evaluated by the new Dutch DV/OEB protein evaluation system. Dry roasting significantly increased BCP, BST, TPSI, ABCP, DVE (p<0.001) and decreased FOM, E_MP, AMP, N_MP and OEB (p<0.001) with increasing temperatures and times except that when temperature was at $110^{\circ}C$. The values of BCP, BST, TPSI, ABCP and DVE at $150^{\circ}C/45min$ for WLS and WFB were increased 2.2, 3.7; -, 2.0; 1.7, 1.7; 2.3, 3.7 and 1.7, 1.7 times and the values of FOM, E_MP, AMP, N_MP and OEB at $150^{\circ}C/45min$ for WLS and WFB were decreased by 15.3, 25.8; 18.1, 25.8; 18.7, 25.8; 54.6, 41.6 and 82.3% 54.7%, respectively, over the raw WLS and WFB. The results indicated that though dry roasting reduced microbial protein synthesis due to reducing FOM, TPSI didn't decrease but highly increased due to increasing BCP more than enough for compensation of the microbial protein decreasing. Therefore the net absorbable DVE in the small intestine was highly increased. The OEB values were significantly reduced for both WLS and WFB but not to the level of negative. It indicated that microbial protein synthesis might not be impaired due to the sufficient N supplied in the rumen, but the high positive OEB values in the most treatments except of $150^{\circ}C$ for 30 and 45 min of WLS (The OEB values: 54.8 and 26.0 g/kg DM) indicated that there were the large amounts of N loss in the rumen. It was concluded that dry roasting at high temperature was effective in shifting protein degradation from rumen to intestines and it increased the DVE values without reaching the negative OEB values. No optimal treatment was found in WLS due to the too high OEB values in all treatments. But dry roasting at $150^{\circ}C$ for 30 and 45 min might be optimal treatments for WLS due to the very lower OEB values.

E1B-19k의 세포내 위치와 Bax와의 Dimerization에 관한 연구 (E1B-19k does not Localize in Mitochondria nor Dimerize Bax even with the Staurosporine)

  • 윤수한;김진영;박승우;안영환;안영민;조기홍;조경기
    • Journal of Korean Neurosurgical Society
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    • 제29권6호
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    • pp.725-730
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    • 2000
  • Purpose : The subcellular localization of E1B-19k has been known cytosol or nuclear membrane by immunohistochemical staining and could dimerize with Bax to regulate cell death also known by the in-vitro immunoprecipitation. We planed to confirm this dimerization of E1B-19k with Bax in vivo in Cos-7 cells by using green fluorescent protein. Material and Method : We cloned E1B-19k and Bax into C3-EGFP. C3-EGFP-E1B-19k, C3-EGFP-Bax, and C3-EGFP-E1B-19k and pcDNA3-Bax were transfected into Cos-7 cells. We explored location of E1B-19k and Bax, and confirmed its dimerization with Bax in transfected living healthy Cos-7 cells by following green fluorescent protein of E1B-19k on the confocal microscope. Results : E1B-19k was located diffusely in cytoplasm and in nucleus but not in mitochondria. It prevented cell death from the apoptosis by staurosporine but its location was not changed. GFP-E1B-19k is not changed its intracellular location with Bax even with staurosporine. Conclusion : These results support that E1B-19k does not localize in mitochondria nor dimerize with Bax even with staurosporine. We could anticipate E1B-19k prevent cell death via the other dimerizing partner or pathways.

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Expression of E. coli Phosphofructokinase Gene in an Autotrophic Bacterium Acidithiobacillus thiooxidans

  • Tian, Ke-Li;Lin, Jian-Qun;Liu, Xiang-Mei;Liu, Ying;Zhang, Chang-Kai;Yan, Wang-Ming
    • Journal of Microbiology and Biotechnology
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    • 제14권1호
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    • pp.56-61
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    • 2004
  • A plasmid pSDK-l containing the Escherichia coli phosphofructokinase-l gene (pfkA) was constructed, and transferred into extremely acidophilic Acidithiobacillus thiooxidans Tt-7 by conjugation with the aid of plasmid RP4 at a frequency of $10^{-5}$ per recipient. This plasmid was stable in A. thiooxidans. The pfkA gene from E. coli could be expressed in this obligately autotrophic bacterium, but the enzyme activity (21.6 U/g protein) was lower than that in E. coli (K12: 85.9 Dig protein; DF1010 carrying plasmid pSDK-l: 96.6 U/g protein). In the presence of glucose, the Tt-7 transconjugants consumed glucose, leading to a better growth yield.

Gel filtration에 의한 콩나물 제조중(製造中) 단백질(蛋白質)의 변화조사(變化調査) (Changes of Protein During Growth of Soybean Sprout)

  • 양차범;박상기;윤석권
    • 한국식품과학회지
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    • 제16권4호
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    • pp.472-474
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    • 1984
  • 대두(大豆)와 대두(大豆)의 발아과정별(發芽過程別) 수추출단백질(水抽出蛋白質)을 Sephadex G-200에 의해서 gel fietration 으로 분획(分劃)한 결과 자엽부(子葉部)에서는 5 개의 분획 (A, B, C, D 및 E) 이 얻어졌고 표품(標品)의 gel filtration과 disc gel electrophoresis 로서 그중(中) A분획이 혼탁물질, B가 11S, C가 7 S, D가 2S로 동정(同定)되었으며 E는 전기영동상에 1 peak를 보였다. 발아(發芽)에 따른 이들 분획(分劃) 변화(變化)는 자명부(子蓂部)에서는 7 S가 먼저 감소되고 2 S가 그 다음이며 11S가 맨나중에 감소되었다. A분획(分劃)은 6 일까지 증가되다가 감소를 보이고 E분획(分劃)은 계속 증가추세를 보이었다. 배축(胚軸)에서는 단지 2 개의 분획(分劃)이 나타났고 그 하나는 B+C(11S+ 7S)의 혼합물이었고 다른 하나는 E분획(分劃)이었다. 발아(發芽)에 따라서 11S+ 7 S 분획(分劃)은 큰 변화(變化)가 없었고 E 분획(分劃)은 약간의 증가를 보였다.

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박하에서 분리한 Caffeic Acid Methyl Ester가 자궁경부암 바이러스 발암단백질 E6의 기능에 미치는 영향 (Effects of Caffeic Acid Methyl Ester from Mentha arvensis Linne var. piperascens on Function of E6 Oncoprotein of HPV 16)

  • 백태웅;이경애;안민정;주혜경;오현철;안종석;조용권;명평근;윤도영
    • 약학회지
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    • 제48권6호
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    • pp.328-334
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    • 2004
  • Cervical cancer is one of the leading causes of female death. Viral oncoproteins E6 and E7 are selectively retained and expressed in carcinoma cells infected with HPV (Human pa pilloma virus) type 16 and cooperated in immotalization and transformation of primary keratinocyte. E6 and E7 oncoproteins interfere the functions of tumor suppressor proteins p53 and retinoblasoma protein (pRb), respectively. Among a lots of natural products, Mentha arvensis Linne var.piperascens have inhibitory effects on bindings between E6 oncoprotein and tumor suppressor p53, E3 ubiqutin- protein ligase (E6AP). HPV oncoprotein inhibitors from Mentha piperita L. were isolated by solvent partition and column chromatography (Silica gel, RP-18) and inhibitory compounds were finally purified by HPLC using an ELISA screening system based on binding between E6 and E6AP. The aim of this study is to identify the structure of inhibitory compounds and to investigate whether these compounds have inhibitory effects on functions of E6 oncoprotein. We investigated whether caffeic acid methyl ester (CAM) extracted from Mentha piperita L. could inhibit the function of E6 oncoprotein. CAM inhibited the in vitro binding of E6 and E6AP which are essential for the binding and degradation of the tumor suppressor p53 and also inhibited the proliferation of human cervical cancer cell lines (SiHa and CaSKi) in a dose response manner. These results suggest that CAM inhibited the function of E6 oncoprotein, suggesting that it can be used as a potential drug for the treatment of cervical cancers infected with HPV.

Purification and Characterization of Cop, a Protein Involved in the Copy Number Control of Plasmid pE194

  • Kwak, Jin-Hwan;Kim, Jung-Ho;Kim, Mu-Yong;Choi, Eung-Chil
    • Archives of Pharmacal Research
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    • 제21권3호
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    • pp.291-297
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    • 1998
  • Cop protein has been overexpressed in Escherichia coli using a T7 RNA polymerase system. Purification to apparent homogeneity was achieved by the sequential chromatography on ion exchange, affinity chromatography, and reverse phase high performance liquid chromatography system. The molecular weight of the purified Cop was estimated as 6.1 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). But the molecular mass of the native state Cop was shown to be 19 kDa by an analytical high performance size exclusion chromatography, suggesting a trimer-like structure in 50 mM Tris-HCI buffer (pH 7.5) containing 100 mM NaCl. Cop protein Was calculated to contain $39.1% {\alpha}-helix, 16.8% {\beta}-sheet$, 17.4% turn, and 26.8% random structure. The DNA binding property of Cop protein expressed in E. coli Was preserved during the expression and purification process. The isoelectric point of Cop was determined to be 9.0. The results of amino acid composition analysis and N-terminal amino acid sequencing of Cop showed that it has the same amino acid composition and N-terminal amino acid sequence as those deduced from its DNA sequence analysis, except for the partial removal of N-terminal methionine residue by methionyl-aminopeptidase in E. coli.

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Production of Toxin Protein by Recombinant Escherichia coli with a Thermally Inducible Expression System

  • Jong, Se-Han;Chang, Ho-Nam;Chang, Yong-Keun;Rhim, Seong-Lyul
    • Journal of Microbiology and Biotechnology
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    • 제6권6호
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    • pp.451-455
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    • 1996
  • Physiological studies on the expression of Bacillus thuringiensis subsp. tenebrionis (Btt) gene coding for insecticidal protein in recombinant Escherichia coli 537 were carried out to identify optimal culture condition. It was necessary to shift culture temperature from 30 to $42^{\circ}C$ to express the gene. Expression of the Btt toxin gene by recombinant E. coli 537 began within one hour after induction. Complex nitrogen sources increased production of the insecticidal protein. The total insecticidal protein was 0.5 g/I when using yeast extract as a complex nitrogen source. Soybean hydrolysate showed apparently the highest induction efficiency. After induction, the cellular content of the insecticidal protein was 5.4 times higher than it had been before induction. The optimal cultivation strategy was found to grow cells for 7hours at $30^{\circ}C$ and then 5-8 hours at $42^{\circ}C$. The optimal cultivation pH for the production of insecticidal protein was 6.5. The Btt toxin produced by the recombinant E. coli 537 was found to have the same level of potency against Colorado potato beetle as the original toxin.

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