• 제목/요약/키워드: inducible promoter

검색결과 193건 처리시간 0.023초

Cloning and expression of human $\beta$$_2$-adrenergic receptor in Saccharomyces cerevisiae

  • 장원진;안진현;고광호;강현삼
    • 한국응용약물학회:학술대회논문집
    • /
    • 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
    • /
    • pp.295-295
    • /
    • 1994
  • The human ${\beta}$$_2$-adrenergic receptor (h${\beta}$$_2$AR) contains seven clusters of hydrophobic amino acids suggestive of membrane-spanning domains and its gene is intronless. The genomic gene encoding h${\beta}$$_2$AR has been isolated by polymerase chain reaction. To express h${\beta}$$_2$AR in Saccharomyces cerevisiae, a modified h${\beta}$$_2$AR gene was fused to signal peptide sequence of Killer toxin gene from Kluyveromyces lactics. This fusion gene was expressed under the galactose-inducible GAL10 promoter. The ligand binding experiments showed that the functional h${\beta}$$_2$AR was expressed at a concentration three times as much as that found in Hamster lung.

  • PDF

Simple Purification of shiga Toxin B Chain from Recombinant Escherichia coli

  • Oh, Young-Phil;Jeong, Seong-Tae;Kim, Dae-Weon;Kim, El-Chae;Yoon, Ki-Hong
    • Journal of Microbiology and Biotechnology
    • /
    • 제12권6호
    • /
    • pp.986-988
    • /
    • 2002
  • A plasmid expression vector of pEStxl encoding a mature form of the B chain of the Shiga toxin was constructed without a signal peptide under the control of an inducible n promoter. The encoded protein was purified to 90% by simple heat treatment, and then further purified to 95% by Phenyl-Sepharose and DEAE-Sepharose chromatographies, all in a single day. Accordingly, this expression system and heat treatment could facilitate the rapid purification of gram-scale amounts of the Shiga toxin B subunit from recombinant Escherichia coli cells.

Gene Expression and Secretion of the Anticoagulant Hirudin in Saccharomyces cerevisiae

  • Sohn, Jung-Hoon;Lee, Sang-Kwon;Choi, Eui-Sung;Rhee, Sang-Ki
    • Journal of Microbiology and Biotechnology
    • /
    • 제1권4호
    • /
    • pp.266-273
    • /
    • 1991
  • Hirudin, a 65-amino acid protein isolated from the salivary gland of the bloodsucking leech, Hirudo medicinalis, is a potent thrombin-specific inhibitor and blocks the thrombin-mediated conversion of fibrinogen to fibrin in clot formation. We have studied the gene expression and secretion of hirudin in yeast. Saccharomyces cerevisiae. A gene coding for hirudin was synthesized based on the amino acid sequence and cloned into a yeast expression vector $YEG{\alpha}-1$ containing the ${\alpha}-mating$ factor pre-pro leader sequence and galactose-inducible promoter, GALl0. Recombinant S. cerevisiae was found to secrete biologically active hirudin into the extracellular medium. The secreted recombinant hirudin was recovered from the culture medium and purified with ultrafiltration and reverse phase high performance liquid chromatography. Approximately 1 mg of hirudin per liter was produced under suboptimal culture conditions and brought to about 90% purity in two steps of purification.

  • PDF

메나놀 자화 효모 Hansenula polymorpha를 이용한 재조합 인체 표피 성장인자 유전자의 발현 및 분비 (Gene Expression and Secretion of Human Epidermal Growth Factor in a Methylotrophic Yeast Hansenula polymorpha)

  • 오용익;손정훈;최의성;김희철;이상기
    • 한국미생물·생명공학회지
    • /
    • 제22권5호
    • /
    • pp.477-484
    • /
    • 1994
  • Using a methylotrophic yeast Hansenula polymorpha, a heterologous gene expression and secretion system was developed for the production of hEGF(human Epidermal Growth Factor) which has been shown to promote epithelial cell proliferation and to inhibit gastric acid secretion. The hEGF gene was chemically synthesized according to the preferred codon usage in H. polymor- pha and expressed under the control of the strong and inducible methanol oxidase(MOX) promoter. The mating factor $\alpha$ pre-pro leader sequence of Saccharomyces cerevisiae was employed for hEGF to be secreted into the extracellular medium. This expression cassette was stably integrated into the host chromosomal DNA. Mature hEGF was efficiently expressed and secreted into the extracel- lular medium. About 24 mg/l of hEGF was detected in the cuture supernatant of a transformant with pA-EGF3 under the suboptimal culture conditions.

  • PDF

Enhanced Gene Expression by Fusion to Rice-ubiquitin in Yeast

  • Kim, Young-Mi
    • Journal of Applied Biological Chemistry
    • /
    • 제43권1호
    • /
    • pp.1-4
    • /
    • 2000
  • Chloramphenicol acetyl CoA transferase (CAT) and angiotensin- converting enzyme inhibitory peptide (ACEI) were fused to C-terminal region of rice ubiquitin to examine the level of transcripts or enzyme activities in yeast. When two chimeric genes under an inducible Gall promoter control were transformed into Saccharomyces cerevisaie, both CAT and ACE inhibitory activities were enhanced by three to four-fold as compared to those containing no ubiquitin gene. However, the levels of transcripts of ubiquitin fused and un fused genes were not significantly different each other. Therefore, it was suggested that the expression of foreign genes was post-transcriptionally enhanced by fusion of plant ubiquitin in heterologous organisms such as yeast.

  • PDF

C/EBP$\beta$ mediated inhibition of PAH-inducible CYPlAl expression by Oltipraz, a cancer chemopreventive agent

  • Cho, Il-Je;Kim, Sang-Geon
    • 대한약학회:학술대회논문집
    • /
    • 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
    • /
    • pp.85.3-86
    • /
    • 2003
  • Oltipraz, a cancer chemopreventive agent, induces CYP1A1 to a certain extent by transactivation of the gene via the Ah receptor (AhR)-xenobiotic response element (XRE) pathway. Previously, we showed that oltipraz promoted CCAAT/enhancer binding protein (C/EBP ) activation, which leads to the induction of glutathione S-transferase. Given that oltipraz activates C/EBP for gene transactivation and that the putative C/CBP binding site is located in CY)1A1 promoter region, this study investigated the effect of oltipraz on CYP1A1 induction by 3-methylcholanthrene (3-MC). (omitted)

  • PDF

포도 VIASR 유전자 프로모터의 분리 및 발현 분석 (Cloning and Expression Analysis of a Grape asr gene, VlASR Containing a Promoter Region.)

  • 길준영;피재호
    • 생명과학회지
    • /
    • 제17권8호통권88호
    • /
    • pp.1157-1165
    • /
    • 2007
  • 포도 ASR (VvMSA) 단백질은 hexose transporter 유전자 VvHT1의 전사를 조절하는 조절 인자 중의 하나로서 sugar 및 abscisic acid (ABA) 신호에 의해 발현이 유도된다. 본 연구진은 ACP RT-PCR (annealing control primer reverse transcriptase-polymerase chain reaction) 방법을 이용하여 포도 과실발달 과정에서 조절되는 유전자 중 VvMSA와 동일한 cDNA (VlASR)를 클로닝하였다. 이 유전자는 착과 시기에 발현되기 시작하여 과실이 발달하면서 점점 증가하여 착과 후 10 주에 가장 많이 발현되며, 숙기 후반에는 도리어 발현양이 감소하였다. 포도 asr 유전자의 조절기작을 밝히기 위해, 이 유전자의 genomic clone을 분리하였다. 총 1375 bp로 이루어진 이 유전자 절편에는 open reading frame과 100 bp의 intron을 포함하고 있다. 약 600 bp 길이의 프로모터 내에는 sugar 신호전달과 연관이 있는 것으로 알려진 sugar box(sucrose box 3 +sucrose response box 1)가 있다. 프로모터 절편을 reporter 유전자와 연결하여 Arabidopsis에 도입하고 형질전환체를 분석한 결과, reporter 유전자는 sucrose 처리와 상관없이 항상 발현되었다. 이러한 결과는 포도에서 보고된 ASR/VvHT1를 매개로 하는 sugar/ABA 신호전달계가 asr 유전자가 없는 Arabidopsis에서는 작동되지 않음을 시사하고 있다.