• Title, Summary, Keyword: Promoter

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Expression and Secretion of Recombinant Inulinase under the Control of GAL or GAP Promoter in Sacharomyces cerevisiae (Sacharomyces cerevisiae에서 GAL또는 GAP 프로모터 조절에 의한 재조합 Inulinase의 발현 및 분비)

  • 남수완;임현정정봉현장용근
    • KSBB Journal
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    • v.11 no.4
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    • pp.445-452
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    • 1996
  • To investigate the promoter effect on heterologous gene expression in S. cerevisiae, the recombinant plasmids pYI11, pYI12, pYI10-2, and pYIGP were constructed to contain the inulinase gene (INUI) as a reporter under the control of GAL10, GAL7, GAL1, and GAP promoters, respectively. When the yeasts transformants were cultivated on galactose-containing rich media, the cell growth reached to 36-39 OD600 at 72 hours of cultivation. The specific growth rates of the cells harboring the four different plasmids decreased similarly : they dropped from $0.24 h^{-1}$ during the glucose-consuming period to 0.04 -$0.10 h^{-1}$ during the galactose-consuming period (gene expression phase for GAL promoter system). After the depletion of glucose, the expression of inulinase gene was started and reached to maximal levels of 4.3(GAL1 promoter), 4.0(GAL10 promoter), 3.8(GAL7 promoter), and 1.6(GAP promoter) unit/mL at 72 hours of cultivation. Based on the maximal expression level and activity staining on the plate, the promoter strength was in the order of GAL1, GAL10, GAL7 and GAP promoter. While the GAL-promoter systems showed a high plasmid stabilities of more than 78%, the GAP-promoter plasmid revealed a lower plasmid stability of 55%. Most of inulinase activity (98%) was found in the extracellular medium, indicating that the secretion efficiency of inulinase is independent on the type of promoter.

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Expression of Developmentally Regulated Promoter of Alkali-tolerant Bacillus sp. YA-I4 (알칼리 내성 Bacillus sp. YA-14에서 유래된 생육단계 조절 promoter의 발현)

  • 박영서;구본탁;박희경;유주현;김진만
    • Microbiology and Biotechnology Letters
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    • v.18 no.4
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    • pp.429-432
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    • 1990
  • The promoter isolated from chromosomal DNA of an alkali-tolerant Bacillus sp. YA-14 was subcloned and biochemically characterized. Also the relationships between the promoter activity and sporulation were investigated. In alkali-tolerant Bacillus sp. and Bacillus subtilis, the activity of promoter began to increase at the onset of sporulation with the same mode, and repressed in the presence of 1.0% (wtv) glucose. Among five spoO genes, three epoO genes (spoOB, spoH, spoOJ) were required for promoter expression.

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Synthetic Regulatory Elements of the Nopaline Synthase Promoter in Higher Plants (고등 식물에서 Nopaline Synthase Promoter의 합성 조절 요소)

  • Kim, Young-Hee
    • Korean Journal of Plant Tissue Culture
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    • v.22 no.4
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    • pp.201-205
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    • 1995
  • The synthetic oligomers called nos right palindrome (RP) element and left palindrome (LP) element were inserted into nos.minimal promoter nos 5'-101 deletion mutant The activity of nos promoter was measured by studying the expression pattern of gene fusion between nos promoter and reporter genes such as chloramphenicol acetyltransferase and $\beta$-glucuconidase. Analysis of transgenic tobacco plane carrying transgene showed that the activity of nos minimal promoter activity was recovered by insertion of synthetic nos RP element. Nos RP element insertion of nos minimal promoter was induced by auxin, dithiothreitol, salicylic acid and methyl jasmonate.

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Expression of \beta-agarase Gene and Carabolite Repression in Escherichia coli by the Promoter of Alginate Lyase Gene Isolated from Marine Pseudomonas sp. (해양의 Pseudomonas sp. 로부터 분리한 alginate lyase 유전자의 promoter에 의한 대장균 내에서의 \beta-agarase 유전자의 발현과 catabolite repression의 변화)

  • 공인수;박제현;한정현;최윤혁;이종희;진철호;이정기
    • Microbiology and Biotechnology Letters
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    • v.29 no.2
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    • pp.72-77
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    • 2001
  • Expression of f3 ~agarase Gene and Catabolite Repression in Escherichia coli by the Promoter of Alginate Lyase Gene Isolated from Marine Pseudomonas sp. Jin, Cheal~Ho, J~Hyeon Park, Jeong-Hyun Han, YoonM Hyeok Chae, Jong~Hee Lee, Jung-Kee Lee!, and In-800 Kong*. Faculty of Food Science and Biotechnology, Pukyong National UniversitYt Pusan 608-737, Korea, llnBioNet Co. 1690-3 Taejon 306-230, Korea - Promoter is a key factor for expression of the recombinant protein. There are many promoters for overexpression of protein in various organisms. The aly promoter of Pseudomonas sp. W7 isolated from marine environment was known to be a constitutive expression promoter of the alginate lyase gene, and it's promoter activity is repressed by glucose in Escherichia coli. To investigate the catabolite repression of the aly promoter ~md association between the promoter mutants, f3 agarase gene, which was also cloned from Pseudomonas sp. W7 was connected to the aly promoter with the sequence the coding 46 N-terminal amino acids ofthe alginate lyase gene. The constructed plasmid was introduced into E. coli and the agarase activity was measured. Fourty six amino acids of the alginate lyase gene was serially deleted using peR to the direction of 5' upstream region and subcloned. The agarase was overexpressed by the aly promoter and the production of agarase was repressed by the addition of glucose into culture media. Fourty six amino acids of alginate lyase did not affect the production of agarase at all. The deletion of a putative stem-loop structure in the aly promoter induced the decrease of f3 -agarase productivity.

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Analysis of Heat Shock Promoters in Hansenula polymorpha: The TPS1 Promoter, a Novel Element for Heterologous Gene Expression

  • Amuel, Carsten;Gellissen, Gerd;Cor;Suckow, Manfred
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.5 no.4
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    • pp.247-252
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    • 2000
  • The strength and regulatory characteristics of the heat-inducible HSA1, HSA2 and TPS1 promoters were compared with those of the well-established, carbon source-regulated FMD promoter in a Hansenula polymorpha-based host system in vivo. In addition, the Saccharomyces cerevisiae-derived ADH1 promoter was analysed. While ADH1 promoter showed to be of poor activity in the foreign host, the strength of the heat shock TPS1 promoter was found to exceed that of the FMD promoter, which at present is considered to be the strongest promoter for driving heterologous gene expression in H. polymorpha.

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Selection of Constitutive Promoter for Exoinulinase Production in Fed-Batch Culture of Recombinant Yeast (재조합 효모의 유가배양에서 Exoinulinase생산을 위한 Promoter의 선별)

  • 김이경;고지현;김연희;김성구;남수완
    • Microbiology and Biotechnology Letters
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    • v.29 no.4
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    • pp.206-211
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    • 2001
  • In order to overexpress constitutively the Kluyveromyces marxianus exoinulinase gene (INUI) in Saccharomyces cerevisiae, four episomal expression systems employing GAPDH, ADHI, PGK and ENOI promoters were constructed as p YIGP aADHI -INU, pPGK-INU, and pENOI- INU plasmids respectively, When S cereviais transformants harboring each plasmid were batchwisely cultivated in the fermentor containing 5% glucose medium no significant differences in the cell growth are observed How- ever the experession level of exoinulinase and plasmid stability showed a strong dependency on the promoter employed. The expression levels of exoinulinase were about 1.70 unit/ml for GAPDH promoter 1.67 unit/ml for PGK promoter, 1.29 unit /ml for ADH1 promoter, and 0.80 unit/ml for ENOl promoter. The plasmid stabilites were maintaines above 80% in all experession systems. except the GAPDH promoter system of 55%, Based on the plas- mid stability and expression level of exoinulinase the ADHl and PGK promoter system were selected for the fed - batch culture to overproduce exoinulinase By the intermittent feeding of yeast extract and glucose, both promoter systems gave the cell concentration of about 30 g-dry cell weight/1 byt the maximal exoinulinase activity of 3.70 unit/ml and plasmid stability of 96% in the ADH1 promoter were higher than those (2.70 unit/ml, 80%) of PGK sys- tem Taking into account the plasmid stability and extended culture time the ADH1 promoter systems would be the most feasible expression systems for the constitutive overproduction of exoinulinase through high cell-density fed- batch cultures using non-selective rich medium.

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Properties of Promoters from Alkali-tolerant Bacillus sp. (알카리 내성 Bacillus속 Promoter의 특성)

  • 유주현;구본탁;박영서;정용준;배동훈;오두환
    • Microbiology and Biotechnology Letters
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    • v.16 no.5
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    • pp.343-347
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    • 1988
  • The promoters of alkali-tolerant Bacillus sp. had been cloned in the promoter probe vector pPL703 and recombinant plasmid p-12 had been constructed. As a result of subcloning, two different promoters were found to exist in the cloned 2.9 kb promoter fragment and two recombinant plasmids p-l2B1 and p-l2B2, each harboring different promoter, were constructed. The promoter activity, which was expressed in the CAT specific activity, of p-l2B1 was 7 times higher than that of p-l2B2. The promoter activity as a function of growth revealed that both promoters of p-l2B1 and p-l2B2 were expressed after the late logarithmic growth phase and repressed in the presence of 1.0% (w/v) glucose.

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Studies on the Development of Yeast Promoter for the Gene Expression (효모(酵母) 유전자(遺傳子) 발현용(發現用) Promoter 개발(開發)에 관(關)한 연구(硏究))

  • Chung, Ho-Kwon;Park, Joon-Hee;Shim, Sang-Kook;Chung, Dong-Hyo
    • Applied Biological Chemistry
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    • v.38 no.1
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    • pp.7-12
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    • 1995
  • The purpose of this study was the development of promoter for the lacZ' gene. Two heterologous promoter I and II of lacZ' gene were isolated from chromosomal DNA Bam HI fragment of yeast. The size of the promoter I was estimated to be 2.5 kb and ${\beta}-galactosidase$ activity was 124.6 U/mg protein, and the size of the promoter II was 4.0 kb and its ${\beta}-galactosidase$ activity was 168.8 U/mg protein, respectively. The stability of the recombinant YEp plasmid in the transformant was from 52.7 to 67.4% at minimal medium. YIp plasmid was constructed from YEp plasmid, and expressed both in E. coli and yeast. The promoter I aid II iso-lated from yeast chromosomal DNA can be used for promoter of plasmid YEp and YIp.

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Structural Characterization of the Regulatory Site in virE Promoter of Agrobacterium tumefaciens pTiA6 Plasmid (Agrobacterium tumefaciens pTiA6 플라스미드의 virE 프로모터내 조절부위의 구조적 특성)

  • 음진성
    • Journal of Plant Biology
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    • v.35 no.2
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    • pp.155-163
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    • 1992
  • To elucidate the regulatory mechanism of virE operon in Agrobacterium tumefaciens pTiA6 plasmid at the molecular level, the regulatory site of virE promoter was determined using truncated virE recombinant plasmids obtained by 5' deletion analysis of virE promoter. The size of deleted nucleotides of p]S201, a functional recombinant plasmid, was found to be about 130 nucleotides from 5'-end of virE promoter. On the other hand the size of deleted nucleotides of p]S301, nonfunctional recombinant plasmid, was identified 263 nucleotides by DNA sequencing. Hence it was thought that the essential site of virE promoter was located between about 130th nucleotide and 263th nucleotide. Since the inverted repeat sequence (AACTTTGCGCTATAGGCAMGTT) is included in this essential site of virE promoter, it could be the first recognition site of the RNA polymerase in virE promoter.omoter.

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Promoter Prediction using Genetic Algorithm (유전자 알고리즘을 이용한 Promoter 예측)

  • 오민경;김창훈;김기봉;공은배;김승목
    • Proceedings of the Korean Information Science Society Conference
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    • pp.12-14
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    • 1999
  • Promoter는 transcript start site 앞부분에 위치하여 RNA polymerase가 높은 친화성을 보이며 바인당하는 DNA상의 특별한 부위로서 여기서부터 DNA transcription이 시작된다. function이나 tissue-specific gene들의 그룹별로 그 promoter들의 특이한 패턴들의 조합을 발견함으로써 Specific한 transcription을 조절하는 것으로 알려져 있어 promoter로 인한 그 gene의 정보를 어느 정도 알 수가 있다. 사람의 housekeeping gene promoter들을 EPD(eukaryotic promoter database)와 EMBL nucleic acid sequence database로부터 수집하여 이것들 간에 의미 있게 나타나는 모든 패턴들을 optimization algorithm으로 알려진 genetic algorithm을 이용해서 찾아보았다.

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