• Title/Summary/Keyword: Recombinant expression

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Recombinant Adeno-Associated Virus Expressing Truncated IK Cytokine Diminishes the Symptoms of Inflammatory Arthritis

  • Choi, Seulgi;Park, Hyelim;Minelko, Marstella;Kim, Eun-Kyung;Cho, Mi-Ra;Nam, Jae-Hwan
    • Journal of Microbiology and Biotechnology
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    • v.27 no.10
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    • pp.1892-1895
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    • 2017
  • IK can downregulate interferon-gamma-induced major histocompatibility complex (MHC) class II expression through the MHC class II transactivator, which suggests that IK can inhibit the interactions between immune cells. We delivered adeno-associated virus serotype 2 (AAV2) encoding the genes for truncated IK (tIK) or green fluorescent protein (GFP) to DBA1/J mice via intravenous injection. Seven weeks after injection, collagen-induced arthritis was induced in the AAV2-treated mice. AAV2-tIK injection reduced the severity of arthritis and the percentage of pathogenic Th17 cells compared with AAV2-GFP injection. These results suggest a novel gene therapy strategy for treatment of inflammatory arthritis.

Ketoprofen ethyl ester에 대해 높은 광학 선택성을 갖는 (R)- 과 (S)-stereospecfic esterase들의 클로닝과 서열분석 및 발현

  • Kim, Ji-Yeon;Choe, Gi-Seop;Kim, Geun-Jung;Yu, Yeon-U
    • 한국생물공학회:학술대회논문집
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    • 2001.11a
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    • pp.625-628
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    • 2001
  • To isolate novel strains that hydrolyzed the rac-ketoprofen ethyl ester to ketoprofen in the stereospecific manner, we screened broad ecological niches and soil samples in which the activity was expected to be found. From thousands of strains, we isolated a Pseudomonas sp. S34 producing a (S)-stereospecific esterase, and a thermostable esterase with (R)-form selectivity was also 。 btained from Bacillus stearothermophilus JYl44. To further analyse the gene structure and to induce a high level expression, two genes from each strain were cloned and sequenced. BLAST search results with the esterase gene from 534 revealed that both of gene (80-84 %) and amino acid sequences (89- 95 %) were highly conserved in the related esterases from Pseudomonas strains (fluorescens and aeruginosa). The thermostable esterase from JY144, however, revealed a relative low homology (45-52 %) to other esterase and/or lipase from related strains. Obviously, a complete conversion with pure enantiomer (R - or S) were readily achieved by recombinant clones expressing either (R)- or (S)- stereospecific esterase.

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Present and Perspective on Insect Biotechnology (곤충생물공학의 현재와 전망)

  • Choi, Hwan-Suk;Kim, Sun-Am;Shin, Hyun-Jae
    • KSBB Journal
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    • v.30 no.6
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    • pp.257-267
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    • 2015
  • Insects are the most successful organisms on earth in terms of their diversity and adaptability. Insect biotechnology using this insect resource is an emerging area for future biotechnology with various applications. Insect resources have long been used to make food and/or functional food, feed, cosmetics as well as medicine and industrial ingredients. Recently, one of the most well-known industrial material from insect is spider silk that could be commercialize in near future. The insect cell lines have been used to express recombinant proteins that were difficult to be functional expression. For public purpose, while, the insect could be good amenity source and plant farming, so leisure resource. Only the interdisciplinary research will guarantee the successful story for insect biotechnology. And biochemical engineers should used insect as a bioresource for new products with applications in medicine, agriculture, and industrial biotechnology in near future. This review will cover state-of-the art of this field and the research and application areas of insect biotechnology and the possible role of biochemical engineer for the development of the future biotechnology using this bioresource.

Large scale purification and characterization of recombinant human autotaxin/lysophospholipase D from mammalian cells

  • Song, Yuanda;Dilger, Emily;Bell, Jessica;Barton, William A.;Fang, Xianjun
    • BMB Reports
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    • v.43 no.8
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    • pp.541-546
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    • 2010
  • We utilized a mammalian expression system to purify and characterize autotaxin (ATX)/lysophospholipase D, an enzyme present in the blood responsible for biosynthesis of lysophosphatidic acid. The human ATX cDNA encoding amino acids 29-915 was cloned downstream of a secretion signal of CD5. At the carboxyl terminus was a thrombin cleavage site followed by the constant domain (Fc) of IgG to facilitate protein purification. The ATX-Fc fusion protein was expressed in HEK293 cells and isolated from conditioned medium of a stable clone by affinity chromatography with Protein A sepharose followed by cleavage with thrombin. The untagged ATX protein was further purified to essential homogeneity by gel filtration chromatography with a yield of approximately 5 mg/liter medium. The purified ATX protein was enzymatically active and biologically functional, offering a useful tool for further biological and structural studies of this important enzyme.

Effects of Kamichihyo-san on Anti-CD40 and Recombinant Interleukin-4 Induced Cytokine Production and Immunoglobulin E in Highly Purified Mouse B Cells (생쥐의 B 세포에서 anti-CD40과 rIL-4로 유도된 사이토카인 생산과 면역글로블린 E에 대한 가미치효산의 효과)

  • Ham Chul in;Park Yang chun
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.17 no.6
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    • pp.1479-1486
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    • 2003
  • In order to evaluate the antiallergic effects of Kamiohihyosan(KCHS), studies were done. We measured the cytotoxic activity for lung fibroblast cell, cytokines transcript expression, production of IL-4, IL-10, IFN-γ, proliferation of B cell in anti-CD40mAb plus rIL-4 stimulated murine splenic B cells. The results were obtained as follows: KCHS was not showed cytotoxicity in the fibroblast lung cell, KCHS increased the gene synthesis of INF-γ, TNF-α, IL1-β, IL-6, IL-10(m-RNA), KCHS decreased the gene synthesis of IL-4, IL-5, TGF-β(m-RNA), KCHS decreased the appearance of IL-4, IgE significantly, KCHS increased the appearance of IL-10, IFN-γ significantly, KCHS decreased the proliferation of B cell significantly, The facts above prove that KCHS is effective against the allergy. Thus, I think that we should study on this continuously.

Molecular cloning and characterization of peroxiredoxin from Toxoplasma gondii

  • Son, Eui-Sun;Song, Kyoung-Ju;Shin, Jong-Chul;Nam, Ho-Woo
    • Parasites, Hosts and Diseases
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    • v.39 no.2
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    • pp.133-141
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    • 2001
  • A cDNA of 1.1 kb comprising the gene encoding the peroxiredoxin of Toxo-plasma gondii(TgPrx) has been cloned. The open reading frame of 591 Up was translated into a protein of 196 amino acids with a molecular mass of 25 kDa. Conserved 2 cysteine domains of Phe-Val-Cys-Pro and Glu-Val-Cys-Pro indicated TgPrx belonged to 2-Cys Prx families. TgPrx showed the highest homology with that of Arabidopsis thaliana by 53.9% followed by Entamoeba histolytica with 39.5% by the amino acid sequence alignment. Polyclonal antibody against recombinant TgPrx detected 25 kDa band in T. gondii without binding to host cell proteins TgPrx was located in the cytoplasm of T. gondii extracellularly or intracellularly by immunofluorescence assay. The expression of TgPrx was increased as early as 30 min after the treatment with artemisinin in the intracellular stage, while no changes in those of host Prx I and TgSOD. This result implies that TgPrx may function as an antioxidant protecting the cell from the attack of reactive oxygen intermediates. It is also suggested that TgPrx is a possible target of chemotherapy.

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Cancer Gene Therapy : Chemosensitization by an Enzyme-Prodrug Activation Strategy

  • Chung, Injae
    • Proceedings of the Korean Society of Applied Pharmacology
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    • 1997.11a
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    • pp.35-39
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    • 1997
  • Recent development of human genetics and techniques of gene transfer and expression have opened the way for investigating novel approaches based on the genetic modification of cells to treat both inherited and acquired diseases. This approach is referred to as gene therapy. Over the past few years, gene therapy has moved from the laboratory to phase I clinical trials. Although the clinical performance of gene transfer experiments is still in an early phase of development, the NIH of Health Recombinant DNA Advisory Comittee (RAC) has approved more than 150 protocols that involve gene transfer or putative gene therapy procedures in clinical settings. Many sectors of society in United States have participated in the design and formulation of these clinical trials through local Institutional Review Boards, the National Institutes of Health (NIH) RAC, the Chemotherapy Evaluation Program of the National Cancer institute, and the FDA. Currently, clinical trials involving gene modification are under way at many medical centers throughout the United Slates. The goals of these trials are as follows. (1) The design should be directed to short-term achievable goals. (2) Each clinical trial is best considered as an intermediate step in a multistep process. (3) The design should identify evaluable proximate endpoints for toxicity and for efficacy, (4) The potential benefits and possible risks for patients participating in these trial should be defined.

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Roles of Neutral Sphingomyelinase 1 on CD95-Mediated Apoptosis in Human Jurkat T Lymphocytes

  • Lee, Hyun-Min;Surh, Bo-Young;Chun, Young-Jin
    • Biomolecules & Therapeutics
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    • v.18 no.3
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    • pp.262-270
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    • 2010
  • CD95 receptor is a member of tumor necrosis factor receptor family that mediates apoptosis in many cell types when bound by CD95 ligand or cross-linked by agonistic anti-CD95 antibodies. To determine the role of neutral sphingomyelinase (nSMase) on CD95-mediatd apoptosis, human Jurkat T lymphocytes were exposed to recombinant human CD95 ligand. Treatment with CD95 ligand induced cell death in a concentration and time-dependent manner. CD95-induced cell death was suppressed by inhibitors of SMase such as AY9944 or desipramine. Transfection with human nSMase1 siRNA plasmid into CD95 ligand-treated cells significantly prevented CD95-mediated cell death. CD95-mediated elevation of intracellular ceramide level detected by FACS analysis with anti-ceramide antibody was also decreased by nSMase1 siRNA. Knock-down of nSMase1 expression also blocked cytochrome c release into cytosol and caspase-3 cleavage in CD95-treated cells. Taken together, these results suggest that nSMase1 may play an important role in CD95-mediated apoptotic cell death in Jurkat T cells.

Molecular Cloning and Characterization of CM Case gene (celC) from Salmonella typhimurium UR

  • Yoo, Ju-Soon;Jung, Youn-Ju;Chung, Soo-Yeol;Lee, Young-Choon;Choi, Yong-Lark
    • Journal of Microbiology
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    • v.42 no.3
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    • pp.205-210
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    • 2004
  • The sequence coding for carboxymethylcellulase (CMCase, CelC) was isolated from the DNA of Salmonella typhimurium URl. Comparison between the deduced amino acid sequence of CelC (368 amino acid residues, Molecular mass 41 kDa) and that of the previously published CMCase revealed that this enzyme belongs to the cellulase family 8 and D. The protein was overproduced in Escherichia coli using T7 expression system, and its activity was confirmed by CMC-SDS-PAGE. When the overexpressed CelC protein was tested on cellulose-type substrates, the recombinant protein is able to degrade cellulose-type substrates, such as CM-cellulose, xylan, avicel, lichenan, and laminarin. Optimal temperature and pH for enzyme activity were found to be 50$^{\circ}C$ and pH 6.5, respectively.

Yeast Elf1 Factor Is Phosphorylated and Interacts with Protein Kinase CK2

  • Kubinski, Konrad;Zielinski, Rafal;Hellman, Ulf;Mazur, Elzbieta;Szyszka, Ryszard
    • BMB Reports
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    • v.39 no.3
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    • pp.311-318
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    • 2006
  • One of the biggest group of proteins influenced by protein kinase CK2 is formed by factors engaged in gene expression. Here we have reported recently identified yeast transcription elongation factor Elf1 as a new substrate for both monomeric and tetrameric forms of CK2. Elf1 serves as a substrate for both the recombinant CK2$\alpha$' ($K_m$ 0.38 ${\mu}M$) and holoenzyme ($K_m$ $0.13\;{\mu}M$). By MALDI-MS we identified the two serine residues at positions 95 and 117 as the most probable in vitro phosphorylation sites. Co-immunoprecypitation experiments show that Elf1 interacts with catalytic ($\alpha$ and $\alpha$') as well as regulatory ($\beta$ and $\beta$') subunits of CK2. These data may help to elucidate the role of protein kinase CK2 and Elf1 in the regulation of transcription elongation.