• Title/Summary/Keyword: overexpression

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Overexpression and Purification of Reverse Transcriptase of Retron EC83 by Changing the Downstream Sequence of the Initiation Codon

  • JEONG , DAE-WON;LIM, DONG-BIN
    • Journal of Microbiology and Biotechnology
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    • v.14 no.6
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    • pp.1280-1285
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    • 2004
  • Retron is a prokaryotic genetic element, producing a short single-stranded DNA covalently linked to RNA (msDNA-RNA) by a reverse transcriptase (RT). In retron EC83, msDNA is further processed at between the 4th and the $5^{th}$ nucleotides, leaving a 79 nucleotide-long single-stranded DNA as a final product. To investigate this site-specific cleavage in msDNA synthesis, we purified the RT protein of retron EC83. Initially, RT ORF was cloned under the tac promoter, but the expression was very poor largely because of poor translation. In order to facilitate translation, the nucleotide sequence for the first nine amino acids was randomized with synonymous codons. This change of downstream sequence of translational initiation codon greatly affected the efficiency of translation. We could isolate clones which greatly increased RT production, and their sequences were compared to those of the low producers. The overproduced protein was purified and was shown to have RT activity.

Overexpression of Escherichia coli Thiol Peroxidase in the Periplasmic Space

  • Kim, Sung-Jin;Cha, Mee-Kyung;Kim, Il-Han;Kim, Ha-Kun
    • Journal of Microbiology and Biotechnology
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    • v.8 no.1
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    • pp.92-95
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    • 1998
  • Overproduction of Escherichia coli thiol peroxidase in the periplasmic space was achieved by locating the appropriate gene on a downstream region of the strong T7 promoter. E. coli strain BL21 carrying the recombinant plasmid pSK-TPX was induced by IPTG, lysed, and analyzed by SDS-polyacrylamide gel electrophoresis. A large amount of the overexpressed thiol peroxidase was located in the periplasmic space. A homogeneous thiol peroxidase was obtained from E. coli osmotic shock fluid by simple one-step gel permeation chromatography.

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Mitochondrial Uncoupling Attenuates Age-Dependent Neurodegeneration in C. elegans

  • Cho, Injeong;Song, Hyun-Ok;Cho, Jeong Hoon
    • Molecules and Cells
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    • v.40 no.11
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    • pp.864-870
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    • 2017
  • The uncoupling protein 4 (ucp-4) gene is involved in age-dependent neurodegeneration in C. elegans. Therefore, we aimed to investigate the mechanism underlying the association between mitochondrial uncoupling and neurodegeneration by examining the effects of uncoupling agents and ucp-4 overexpression in C. elegans. Treatment with either DNP or CCCP improved neuronal defects in wild type during aging. Uncoupling agents also restored neuronal phenotypes of ucp-4 mutants to those exhibited by wild type, while ucp-4 overexpression attenuated the severity of age-dependent neurodegeneration. Neuronal improvements were further associated with reductions in mitochondrial membrane potentials. However, these age-dependent neuroprotective effects were limited in mitophagy-deficient mutant, pink-1, background. These results suggest that membrane uncoupling can attenuate age-dependent neurodegeneration by stimulating mitophagy.

Cloning and Overexpression of Methylcatechol 2, 3-Dioxygenase Gene from Toluene-Degrading Pseudomonas putida mt-2(pWWO)

  • Lee, Jeong-Rai;Min, Kyung-Rak;Kim, Young-Soo
    • Archives of Pharmacal Research
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    • v.15 no.4
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    • pp.360-364
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    • 1992
  • Methylcatechol 2, 3-dioxygenase encoded in pWWO megaplasmid of Pseudomonas putida mt-2 has been cloned and overexpressed in Escherichia coli. This enzyme gene has been localized inside 2. 3-kb XhoI fragment derived from the pWWO megaplasmid. Analysis of enzyme activity and SDS-PAGE showed that the cloned methylcatechol 2, 3-dioxygenase gene in E. coli was about 100 fold overexpressed compared with the parental gene in P. putida mt-2 (pWWO). The cloned enzyme exhibited higher ring-fission activity to catechol than catechol derivatives including 3-methylcatechol, 4-methylcatechol, and 4-chlorocatechol.

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Activation of Hypoxia Inducible Factor-1 Alpha by Estrogen Receptor Alpha (에스트로젠 수용체알파에 의한 Hypoxia Inducible Factor-1의 전사 활성조절)

  • Ryu, Kwang-Hee;Lee, Young-Joo
    • YAKHAK HOEJI
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    • v.54 no.2
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    • pp.102-105
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    • 2010
  • Our previous results showed that hypoxia inducible factor-1 (HIF-1) activated estrogen receptor (ER) in the absence of ligand. In this study, we have studied the effect ER overexpression on the activation of HIF-1. ER overexpression induced transcription activation of hypoxia response element driven luciferase and vascular endothelial growth factor. As a negative control, the effect of ER on androgen receptor response element was used. Our result indicate that the two ER$\alpha$ and HIF-1 signaling pathways shares part of the activation pathway.

The Preventive Effects of Bcl-2 and $Bcl-_{XL}$ on Lovastatin-induced Apoptosis of C6 Glial Cells

  • Choi, Jae-Won;Lee, Jong-Min;Oh, Young-Jun
    • The Korean Journal of Physiology and Pharmacology
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    • v.6 no.5
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    • pp.235-239
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    • 2002
  • It has been reported that lovastatin induced cell death and suppressed proliferation in various cell lines. In this study, we examined whether the cytotoxic effects of lovastatin could be prevented by Bcl-2 or $Bcl-_{XL}$ in C6 glial cells. Overexpression of human Bcl-2 or $Bcl-_{XL}$ prevented lovastatin $(25{\mu}M)-induced$ changes such as DNA fragmentation, chromatin condensation, disruption of cell membrane, and cleavage of poly (ADP-ribose) polymerase. Lovastatin-induced inhibition of cell proliferation was unaffected by Bcl-2 or $Bcl-_{XL}$ overexpression. These results suggest that Bcl-2 and $Bcl-_{XL}$ can prevent lovastatin-induced apoptosis in C6 glial cells, though the inhibition of proliferation remains unaffected by these proteins.

NMMP1, a Matrix Metalloprotease in Nicotiana benthamiana Has a Role in Protection against Bacterial Infection

  • Kang, So-Ra;Oh, Sang-Keun;Kim, Jong-Joo;Choi, Do-Il;Baek, Kwang-Hyun
    • The Plant Pathology Journal
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    • v.26 no.4
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    • pp.402-408
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    • 2010
  • Plant matrix metalloproteases (MMPs) are a family of apoplastic metalloproteases closely related to human matrilysins. Up-regulation of Nicotiana benthamiana matrix metalloprotease 1 (NMMP1) expression by treatment with pathogens, ethephon and aging indicates that the gene is related to plant defense and the aging process through ethylene signaling. NMMP1 expression was higher than in normal growth leaves following infection with an incompatible pathogen Pseudomonas syringae pv. tomato T1 or a compatible pathogen P. syringae pv. tabaci and in aged leaves. Transient overexpression of NMMP1 in N. benthamiana leaves lowered the growth of P. syringae pv. tabaci. However, NMMP1-silenced leaves showed increased growth of P. syringae pv. tabaci. These data strongly suggest that NMMP1 in N. benthamiana is a defense related gene, which is positively regulated by ethylene.