• Title, Summary, Keyword: transgenic plant cell cultures

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A Novel Oxidative Stress-inducible Peroxidase Promoter and Its Applications to Production of Pharmaceutical Proteins in Transgenic Cell Cultures

  • Lee, Ok-Sun;Park, Sun-Mi;Kwon, Suk-Yoon;Lee, Haeng-Soon;Kim, Kee-Yeun;Kim, Jae-Whune;Kwak, Sang-Soo
    • Journal of Plant Biotechnology
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    • v.4 no.4
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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.

Production of biopharmaceuticals in transgenic plant cell suspension cultures (형질전환 식물세포배양을 이용한 바이오의약품 생산)

  • Kwon, Jun-Young;Cheon, Su-Hwan;Lee, Hye-Ran;Han, Ji-Yeon;Kim, Dong-Il
    • Journal of Plant Biotechnology
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    • v.36 no.4
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    • pp.309-319
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    • 2009
  • Transgenic plant cell cultures for the production of biopharmaceuticals including monoclonal antibodies, recombinant proteins have been regarded as an alternative platform in addition to traditional microbial fermentation and mammalian cell cultures. Plant-made pharmaceuticals (PMPs) have several advantages such as safety, cost-effectiveness, scalability and possibility of complex post-translational modifications. Increasing demand for the quantity and diversity of pharmaceutical proteins may accelerate the industrialization of PMP technology. Up to date, there is no plant-made recombinant protein approved by USFDA (Food and Drug Administration) for human therapeutic uses due to the technological bottlenecks of low expression level and slight differences in glycosylation. Regarding expression levels, it is possible to improve the productivity by using stronger promoter and optimizing culture processes. In terms of glycosylation, humanization has been attempted in many ways to reduce immune responses and to enhance the efficacy as well as stability. In this review article, all these respects of transgenic plant cell cultures were summarized. In addition, we also discuss the general characteristics of plant cell suspension cultures related with bioreactor design and operation to achieve high productivity in large scale which could be a key to successful commercialization of PMPs.

Enhanced Production of hGM-CSF by Immobilized Transgenic Plant Cell Cultures (형질전환된 식물세포에서 고정화 방법을 통한 hCM-CSF의 생산성 증대 연구)

  • Noha, Yun-Sook;Nama, Hyung-Jin;Choi, Hong-Yeol;Tak, Sa-Ra;Kim, Dong-Il
    • KSBB Journal
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    • v.30 no.2
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    • pp.82-90
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    • 2015
  • Plant cell immobilization can protect plant cells from shear forces and increase the stability of gene. An additional advantage of immobilization is the easiness for performing continuous culture with cell recycling. Therefore plant cell immobilization can overcome the limitations of plant cell applications. In addition, target protein should be selected from pharmaceutical proteins to get rid of low expression level problem. The enhanced production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was investigated in immobilized Nicotiana tabacum suspension cell cultures. When the cells were immobilized in polyurethane foam, specific production of hGM-CSF was higher than that in alginate bead immobilization. Optimum continuous culture condition was the addition of 60 g/L sucrose in growth media with exchanging media every 6 day. Under the same condition, specific hGM-CSF production was 7 times higher in a 500-mL spinner flask than that in 100-mL Erlenmeyer flasks. Therefore, development of an effective immobilization process would be possible when the advantage of easy cell recycling was used. Consequently, enhanced production of target proteins could be possible in immobilized continuous cultures when the advantages of immobilization were applied.

Effects of Antioxidants on Cell Viability and hGM-CSF Production by Transgenic Nicotiana tabacum Suspension Cultures (형질전환된 Nucotiana tabacum 현탁세포배양에서 항산화제가 세포생존도 및 hGM-CSF 생산에 미치는 영향)

  • Kim Yong Hoon;Lee Sang Yoon;Kim Dong Il
    • KSBB Journal
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    • v.19 no.5
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    • pp.374-380
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    • 2004
  • Production of therapeutic proteins by transgenic plant cell suspension cultures is an attractive system alternative to the other expression system. However, plant cell cultures have shown low expression level of foreign proteins and decreased cell viability by the changes of culture conditions. Therefore, it is necessary to enhance cell viability during the culture period. In this study, a quantitative analysis technique was designed to measure relative cell viability for plant suspension cells which have cell wall and aggregates. It was found that the programmed cell death of plant cells by apoptosis was essentially linked with the apoptotic pathway of animal cells. Therefore, effects of nicotinamide, 3-aminobenzamide and antioxidants on cell viability and apoptosis were examined in transgenic Nicotiana tabacum cells producing hGM-CSF. With those additives, cell viability could be maintained and apoptosis could be redued. In the result, the extracellular production of hGM-CSF could be enhanced 2.5 fold. It was also found that the supplementation of glutathione and ascorbic acid suppressed both the cold stress-induced decrease in cell viability and the increase of total genomic DNA fragmentation.

Regulation of γ-Aminobutyric Acid Production in Tobacco Plants by Expressing a Mutant Calmodulin Gene

  • Oh, Suk-Heung;Cha, Youn-Soo
    • Journal of Applied Biological Chemistry
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    • v.43 no.2
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    • pp.69-73
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    • 2000
  • In order to understand the biological role of calmodulin in plants, transgenic plants expressing a mutant calmodulin (VU-4, Iys to ile-115) have been analyzed. We found that tobacco plants expressing VU-4 calmodulin have approximately twofold higher $\gamma$-aminobutyric acid (GABA) levels than the control plants. Cell suspension cultures established from the stem explants of the transgenic tobacco seedlings also have higher levels of GABA than the control cell cultures. Specific activity of glutamate decarboxylase (GAD), which catalyzes the decarboxylation of glutamate to $CO_2$ and GABA, of the transgenic tobacco cell extracts was about twofold higher than the activity of the control cell extracts. Western-blot analysis showed that the GAD is highly expressed in the transgenic tobacco plants. GAD partially purified from tobacco cell extracts showed approximately threefold $Ca^{2+}$/calmodulin-dependent activation. These data suggest that GABA synthesis in the transgenic tobacco plants is elevated, possibly due to higher levels of the calmodulin-dependent GAD enzyme and/or as a result of enhanced activation due to increased levels of the foreign calmodulin.

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Effects of Silkworm Hemolymph on Cell Viability and hCTLA4Ig Production in Transgenic Rice Cell Suspension Cultures

  • Cheon, Su-Hwan;Lee, Kyoung-Hoon;Kwon, Jun-Young;Ryu, Hyun-Nam;Yu, Da-Hyun;Choi, Yong-Soo;Kim, Dong-Il
    • Journal of Microbiology and Biotechnology
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    • v.17 no.12
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    • pp.1944-1948
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    • 2007
  • Silkworm hemolymph (SH), prepared from fifth-instar larvae of Bombyx mori and heat-treated at $60^{\circ}C$ for 30 min, was used to improve cell viability and the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) in transgenic Oryza sativa L. cell suspension cultures. Even though SH could not elevate cell viability at the concentrations up to 3% (v/v), addition of 0.3% (v/v) SH to a culture medium enhanced the production of hCTLA4Ig by 36.8% over an SH-free medium. Moreover, the production period of hCTLA4Ig could be shortened in a 0.3% (v/v) SH-added medium compared with that in an SH-free culture. As a result, addition of 0.3% (v/v) SH improved the productivity of hCTLA4Ig significantly in transgenic rice cell cultures.

High-yield Production of Functional Human Lactoferrin in Transgenic Cell Cultures of Siberian Ginseng(Acanthopanax senticosus)

  • Jo, Seung-Hyun;Kwon, Suk-Yoon;Park, Doo-Sang;Yang, Kyoung-Sil;Kim, Jae-Whune;Lee, Ki-Teak;Kwak, Sang-Soo;Lee, Haeng-Soon
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.11 no.5
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    • pp.442-448
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    • 2006
  • Human lactoferrin (hLf) is an iron-binding glycoprotein that has been considered to play many biological roles in the human, including the stimulation of the immune system, antimicrobial and anti-inflammatory effects, and regulation of iron absorption. We generated transgenic Siberian ginseng (Acanthopanax senticosus) cell cultures producing a functional hLf protein using the signal peptide sequence from the endoplasmic reticulum and driven by an oxidative stress-inducible SWPA2 promoter which is highly expressed in plant cell cultures. The production of hLf increased proportionally to cell growth and showed a maximal level (up to 3.6% of total soluble protein) at the stationary phase in suspension cultures. Full-length hLf protein was identified by immunoblot analysis in transgenic cell cultures of Siberian ginseng. Recombinant hLf (rhLf) was purified from suspension cells of Siberian ginseng by ammonium sulfate precipitation, cation-exchange and gel filtration chromatography. N-terminal sequences of rhLf were identical to native hLf (nhLf). The overall monosaccharide composition of rhLf showed the presence of plant specific xylose while sialic acid is absent. Antibacterial activity of purified rhLf was higher than that of nhLf. Taken together, we anticipate that medicinal Siberian ginseng cultured cells, as demonstrated by this study, will be a biotechnologically useful source for commercial production of functional hLf not requiring further purification.

Enhanced Production of hCTLA4Ig by Suppressing Cell Death in Transgenic Rice Cell Suspension Cultures (형질전환 벼 현탁세포 배양에서 세포 사멸 억제를 통한 hCTLA4Ig 생산성 증대)

  • Kim, Myong-Sik;Nam, Hyung-Jin;Kim, Min-Sub;Kwon, Jun-Young;Kim, Dong-Il
    • KSBB Journal
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    • v.28 no.4
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    • pp.260-268
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    • 2013
  • Transgenic plant cell cultures are an attractive expression system for the production of industrial and pharmaceutical proteins because of their advantages in safety and low production cost. Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was produced and secreted when sugar was depleted in culture medium by transgenic rice cell lines (Oryza sativa L.) using RAmy3D promoter. Due to the production of the target protein by sugar depletion, concomitant occurrence of cell death is inevitable. For that reason, inhibition of cell death for enhancing productivity was necessary for the production period without energy sources. Supplementation of 0.1 mM sodium nitroprusside improved cell viability by 1.4-fold and maximum hCTLA4Ig production by 1.3-fold compared to those of control. Addition of 1 and 10 mM glutathione, N-acetylcysteine (NAC), and nicotinamide inhibited apoptotic-like programmed cell death by decreasing the activity of reactive oxygen species. Production hCTLA4Ig was enhanced 1.4-, 1.25-, and 1.15-fold with 10 mM NAC, 1 mM NAC, and 1 mM glutathione, respectively. In addition, it was found that the supplementation of NAC enhanced the cell viability.

Enhanced Delivery of siRNA Complexes by Sonoporation in Transgenic Rice Cell Suspension Cultures

  • Cheon, Su-Hwan;Lee, Kyoung-Hoon;Kwon, Jun-Young;Choi, Sung-Hun;Song, Mi-Na;Kim, Dong-II
    • Journal of Microbiology and Biotechnology
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    • v.19 no.8
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    • pp.781-786
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    • 2009
  • Small interfering synthetic double-stranded RNA (siRNA) was applied to suppress the expression of the human cytotoxic-T-Iymphocyte antigen 4-immunoglobulin (hCTLA4Ig) gene transformed in transgenic rice cell cultures. The sequence of the 21-nucleotide siRNA was deliberately designed and synthesized with overhangs to inactivate the expression of hCTLA4Ig. The chemically synthesized siRNA duplex was combined with polyethyleneimine (PEl) at a mass ratio of 1:10 (0.33 ${\mu}g$ siRNA:3.3 ${\mu}g$ PEl) to produce complexes. The siRNA complexes (siRNA+PEI) were labeled with Cy3 in order to subsequently confirm the delivery by fluorescent microscopy. In addition, the cells were treated with sonoporation at 40 kHz and 419W for 90 s to improve the delivery. The siRNA complexes alone inhibited the expression of hCTLA4Ig to 45% compared with control. The siRNA complexes delivered with sonoporation downregulated the production of hCTLA4Ig to 73%. Therefore, we concluded that the delivery of siRNA complexes into plant cells could be enhanced successfully by sonoporation.

Production of hGM-CSF from Cell Suspension Culture of Transformed Lettuce Using Agrobacterium-mediated Transformation System (Agrobacterium을 이용한 형질전환 상추의 세포 현탁배양으로부터 hGM-CSF의 생산)

  • Kim, Young-Sook;Kim, Mi-Young;Kwon, Tae-Ho;Yang, Moon-Sik
    • Journal of Plant Biotechnology
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    • v.30 no.1
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    • pp.97-102
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    • 2003
  • Lettuce (Lactuca sativa) was transformed with Agrobacterium tumefacience LBA4404 containing human granulocyte macrophage colony stimulating factor (hGM-CSF) gene to produce in cell suspension cultures. Cell suspension culture was established using callus from transgenic lettuce plant. Integration of hGM-CSF gene into plant chromosome was confirmed through genomic PCR and Southern blot analysis. In addition, Northern blot analysis indicated the expression of the introduced hGM-CSF gene in transformed lettuce. The recombinant hGM-CSF was expressed in transgenic cell cultures derived from transgenic plants as a yield of about 149.0 $\mu\textrm{g}$/L in culture filtrate, which was determined by ELISA. These results demonstrated that transformed lettuce cell suspension cultures could be used as a production system of therapeutic proteins such as hGM-CSF.