• 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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Core Promoter Mutation of ntC1731T and G1806A of Hepatitis B Virus Increases HBV Gene Expression (B형 간염 바이러스의 ntC1731T 및 G1806A의 core 프로모터 돌연변이에 의한 HBV 유전자 발현 증가 분석)

  • Cho, Ja Young;Yi, Yi Kyaw;Seong, Mi So;Cheong, JaeHun
    • Journal of Life Science
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    • v.32 no.2
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    • pp.94-100
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    • 2022
  • Chronic infection by hepatitis B virus (HBV) greatly increases the risk for liver cirrhosis and hepatocellular carcinoma (HCC). The outcome of HBV infection is shaped by the complex interplay of the mode of transmission, host genetic factors, viral genotype, adaptive mutations, and environmental factors. The pregenomic RNA transcription of HBV for their replication is regulated by the core promoter activation. Core promoter mutations have been the reason for acute liver failure and are associated with HCC development. We obtained HBV genes from a patient in Myanmar who was infected with HBV and identified gene variations in the core promoter region. For measuring the relative transactivation activity of the core promoter, we prepared the core-promoter reporter construct. Among the gene variations of the core promoter, the mutations of C1731T and G1806A were associated with increase in the transactivation of the HBV core promoter. Through computer analysis for searching for a tentative transcription factor binding site, we showed that the mutations of C1713T and G1806A newly created C/EBPβ and XBP1-responsive elements of the core promoter, respectively. The ectopic expression of C/EBPβ largely increased the HBV core promoter containing the C1713T mutation and that of XBP1 activated the M95 promoter containing the G1806A mutation. Our efforts to treat and prevent HBV infections are hampered by the emergence of drug-resistant mutations and vaccine-escape mutations. Our results provide the biological properties and clinical significance of specific HBV core promoter mutations.

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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Characterization of an Oxygen-Dependent Inducible Promoter Systems, the nar Promoter of Escherichia coli, and Gram negative host strains

  • Lee, Gil-Ho;Jo, Mu-Hwan;Lee, Jong-Won
    • 한국생물공학회:학술대회논문집
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    • 2001.11a
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    • pp.762-766
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    • 2001
  • The nar promoter of Escherichia coli was known to induce maximally under anaerobic or microaerobic conditions in the presence of nitrate. In this study, the nar promoter was tested to see whether the expression level of a reporter gene which fused lacZ gene at nar promoter's downstream, in the some gram negative host strains(Agrobacterium, Pseudomonas and Rhizobium). A nar promoter system(Combination of nar promoter and gram negative strain) was grown under aerobic conditions to absorbance at 600 nm of nearly 2.0 and then, the nar promoter was induced by lowering DO to 1-2% with alternating microaerobic and aerobic condition in the fermentor cultures, using different gram negative hosts. For a wild type nar promoter (pNW61), it was possible to maintain production of ${\beta}-galactosidase$ activity per cell(specific ${\beta}-galactosidase$ activity) at 14,000, 9600, 45 Miller units in the presence of 1% nitrate. and for a nitrate - independent nar promoter (pNW618) at 12,000, 10,400 and 58 Miller units in the absence of nitrate ion, respectively.

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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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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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