• Journal of Microbiology and Biotechnology
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• 제19권9호
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• pp.966-971
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• 2009

Peroxidase from Coprinus cinereus (CiP) has attracted attention for its high specific activity and broad substrate spectrum compared with other peroxidases. In this study, the functional expression of this peroxidase was successfully achieved in the methylotrophic yeast Pichia pastoris. The expression level of CiP was increased by varying the microbial hosts and the expression promoters. Since a signal sequence, such as the alpha mating factor of Saccharomyces cerevisiae, was placed preceding the cDNA of the CiP coding gene, expressed recombinant CiP (rCiP) was secreted into the culture broth. The Mut Pichia pastoris host showed a 3-fold higher peroxidase activity, as well as 2-fold higher growth rate, compared with the $Mut^s $ Pichia pastoris host. Furthermore, the AOX1 promoter facilitated a 5-fold higher expression of rCiP than did the GAP promoter.

• Journal of Plant Biotechnology
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• 제4권4호
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• pp.143-150
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• 2002

A strong oxidative stress-inducible peroxidase promoter (referred to as SWPA2 promoter) was cloned from tell cultures of sweetpotato (Ipomoea batatas) and characterized in transgenic tobacco cultured cells in terms of biotechnological applications. Employing a transient expression assay in tobacco protoplasts, with five different 5'-deletion mutants of the SWPA2 promoter fused to the $\beta$-glucuronidase (GUS) reporter gene, the 1314 bp deletion mutant showed approximately 30 times higher GUS expression than the CaMV 35S promoter. The expression of GUS activity in suspension cultures of transgenic cells derived from transgenic tobacco leaves containing the -1314 bp SWPA2 promoter-GUS fusion was strongly expressed following 15 days of subculture compared to other deletion mutants, suggesting that the 1314 bp SWPA2 promoter will be biotechnologically useful for the development of transgenic cell lines engineered to produce key pharmaceutical proteins. In this respect, we developed transgenic cell lines such as tobacco (Nicotiana tabacum L. BY-2), ginseng (Panax ginseng) and Siberian ginseng (Acanthopanax senticosus) using a SWPA2 promoter to produce a human lactoferrin (hLf) and characterized the hLf production in cultured cells. The hLf production monitored by ELISA analysis in transgenic BY-2 cells was directly increased proportional to cell growth and reached a maximal level (up to 4.3% of total soluble protein) at the stationary phase in suspension cultures. The SWPA2 promoter should result in higher productivity and increased applications of plant cultured cells for the production of high-value recombinant proteins.

• Journal of Microbiology and Biotechnology
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• 제8권1호
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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.

• BMB Reports
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• 제32권1호
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• pp.51-59
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• 1999

The Korean radish cationic peroxidase (KRCP) promoter, comprising nucleotides -471 to +704 relative to the transcriptional initiation site, was fused to the GUS gene and transformed to tobacco BY-2 cells. We examined how auxin (2,4-dichlorophenoxyacetic acid, 2,4-D), cytokinin (6-benzylaminopurine, BAP), gibberellic acid ($GA_3$), abscisic acid (ABA), methyl jasmonate (MeJA), and phosphatidic acid (PA) affect the GUS expression in the presence or absence of 2,4-D in a modified LS medium. Exogenous 2,4-D or BAP greatly decreased the GUS expression regulated by the KRCP promoter in a modified LS medium containing 0.2 mg/l 2,4-D. $GA_3$ increased the GUS expression and ABA completely reduced the inductive effect of $GA_3$. The GUS expression was also increased dose-dependently by plant defense regulators, MeJA and PA. In contrast to the above results, auxin deprivation from the modified LS medium increased the GUS expression after treatment with exogenous 2,4-D whereas BAP still greatly decreased the GUS expression dose-dependently. $GA_3$ or MeJA slightly decreased the GUS expression. The data suggest that auxin deprivation changes the sensitivity of the suspension cells to exogenous chemicals and that the regulation of the KRCP promoter by 2,4-D, $GA_3$, and MeJA is dependent on auxin, whereas the regulation by BAP is not. This study will be valuable for understanding the function and expression mode of the Korean radish cationic peroxidase in Korean radish.

• BMB Reports
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• 제29권1호
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• pp.52-57
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• 1996

A specific DNA fragment from Korean radish (Raphanus sativus L.) was amplified by performing PCR with oligonucleotide primers which correspond to the highly conserved regions of plant peroxidases. The size of the PCR product was ca. 400 bp, as expected from the known plant peroxidase genes. Comparison of the nucleotide and deduced amino acid sequences of the PCR product to those of other plant peroxidase-encoding genes revealed that the amplified fragment corresponded to the highly conserved region I and III of plant peroxidases. By screening a genomic library of Korean radish using the amplified fragment as a probe, two positive clones, named prxK1 and prxK2, were isolated. Restriction mapping studies indicated that the 5.2 kb Sail fragment of the prxK1 clone and the 4.0 kb EcoRI fragment of the prxK2 clone encode separate isoperoxidase genes. Analyses of the promoter region of the prxK1 clone shows that putative CAAT box, CMT box, and TGA1b binding sequence (5' TGACGT) are present 718 bp upstream from the start codon.

• 한국식물생명공학회:학술대회논문집
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• 한국식물생명공학회 2004년도 춘계학술대회
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• pp.59-59
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• 2004

• Molecules and Cells
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• 제39권8호
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• pp.631-638
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• 2016

Glutathione peroxidase 3 (GPx3), an antioxidant enzyme, acts as a modulator of redox signaling, has immunomodulatory function, and catalyzes the detoxification of reactive oxygen species (ROS). GPx3 has been identified as a tumor suppressor in many cancers. Although hyper-methylation of the GPx3 promoter has been shown to down-regulate its expression, other mechanisms by which GPx3 expression is regulated have not been reported. The aim of this study was to further elucidate the mechanisms of GPx3 regulation. GPx3 gene analysis predicted the presence of ten glucocorticoid response elements (GREs) on the GPx3 gene. This result prompted us to investigate whether GPx3 expression is regulated by the glucocorticoid receptor (GR), which is implicated in tumor response to chemotherapy. The corticosteroid dexamethasone (Dex) was used to examine the possible relationship between GR and GPx3 expression. Dex significantly induced GPx3 expression in H1299, H1650, and H1975 cell lines, which exhibit low levels of GPx3 expression under normal conditions. The results of EMSA and ChIP-PCR suggest that GR binds directly to GRE 6 and 7, both of which are located near the GPx3 promoter. Assessment of GPx3 transcription efficiency using a luciferase reporter system showed that blocking formation of the GR-GRE complexes reduced luciferase activity by 7-8-fold. Suppression of GR expression by siRNA transfection also induced down-regulation of GPx3. These data indicate that GPx3 expression can be regulated independently via epigenetic or GR-mediated mechanisms in lung cancer cells, and suggest that GPx3 could potentiate glucocorticoid (GC)-mediated anti-infla-mmatory signaling in lung cancer cells.

• 한국식물생명공학회:학술대회논문집
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• 한국식물생명공학회 2002년도 추계학술대회
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• pp.49-58
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• 2002

Oxidative stress derived from reactive oxygen species (ROS) is one of the major damaging factors in plants exposed to environmental stress. In order to develop the platform technology to solve the global food and environmental problems in the 21s1 century, we focus on the understanding of the antioxidative mechanism in plant cells, the development of oxidative stress-inducible antioxidant genes, and the development of transgenic plants with enhanced tolerance to stress. In this report, we describe our recent results on industrial transgenic plants by the gene manipulation of antioxidant enzymes. Transgenic tobacco plants expressing both superoxide dismutase (SOD) and ascorbate peroxidase (APX) in chloroplasts were developed and were evaluated their protection effects against stresses, suggesting that simultaneous overexpression of both SOD and APX in chloroplasts has synergistic effects to overcome the oxidative stress under unfavorable environments. Transgenic tobacco plants expressing a human dehydroascorbate reductase gene in chloroplasts were showed the protection against the oxidative stress in plants. Transgenic cucumber plants expressing high level of SOD in fruits were successfully generated to use the functional cosmetic purpose as a plant bioreactor. In addition, we developed a strong oxidative stress-inducible peroxidase promoter, SWPA2 from sweetpotato (Ipomoea batatas). We anticipate that SWPA2 promoter will be biotechnologically useful for the development of transgenic plants with enhanced tolerance to environmental stress and particularly transgenic cell lines engineered to produce key pharmaceutical proteins.

• 한국양식학회지
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• 제10권1호
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• pp.33-37
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• 1997

미꾸라지를 대상으로 어류 유전자 이식을 위한 유용 model system으로 개발하기 위한 연구의 일환으로 잉어의$\beta$-actin 유전자의 promoter와 CAT 유전자가 융합되어 있는 외래 유전자 (pFV4CAT)를 미꾸라지에 이식, transgenic founder 미꾸라지를 생산하였다. PCR 분석결과, 이식된 외래 유전자는 부화 후 9개월된 성어에서 7.4-37.0%의 빈도로 존재하였으며 Southern blot 분석에 의해서 염색체 상의 삽입 가능성을 나타내었다. In situ immunohistochemical analysis 결과 이식된 외래 유전자는 미꾸라지 세포내에서 비교적 높은 빈도로 발현하였으며 그 발현 양상은 개체마다 다양하게 나타났다.

• Journal of Plant Biotechnology
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• 제29권2호
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• pp.69-77
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• 2002

각종 환경스트레스에 의해 생체 내에서 과량으로 생성되는 독성의 활성산소종 (ROS)은 산화스트레스를 유발시켜 식물의 질병, 노화 및 세포사멸을 촉진시킨다. 연구팀은 21세기 당면한 지구규모의 환경, 식량 및 보건문제 해결에 기여할 수 있는 기반기술 (plateform technology)를 개발하기 위하여 식물세포의 항산화기구 규명. 산화스트레스 유도성 항산화효소 유전자 개발. 스트레스 내성식물 개발에 관한 연구를 수행하고 있다. 여기에서는 항산화효소 유전자를 이용한 산업용식물체 개발에 관한 연구팀의 최근 연구결과를 중심으로 소개하였다. SOD와 APX 유전자를 엽록체에 동시에 발현시킨 담배 식물체는 MV, 건조 등 여러 스트레스에 대한 내성을 나타내어, 복합 스트레스내성 농작물개발에 활용이 기대된다 인체 DHAR 유전자를 엽록체에 도입시킨 담배식물체는 정상적으로 DHAR 유전자를 발현시켰으며, MV 등 여러 스트레스에 대한 내성을 나타내었다. 피부 노화방지 등에 관여하여 ROS를 제거하는 SOD를 과실에 과발현시킨 형질전환오이를 성공적으로 개발하여. SOD 오이는 기능성화장품의 용도로 제품개발이 기대된다. 또한 고구마에서 산화스트레스에 특이적으로 발현하는 POD (SWPA2.) promoter를 개발하였다. SWPA2 promoter는 스트레스내성 및 의료용 단백질 등 고부가가치 생리활성물질을 생산할 수 있는 산업용 형질전환 식물체 및 배양세포주 개발에 이용될 수 있을 것으로 기대된다.