• Title/Summary/Keyword: hGM -CSF

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The Effects of Sucrose and Inoculum Size on the Production of hGM-CSF from Plant Cell Culture (식물세포배양에서 당과 식물세포의 농도가 hGM-CSF의 생산에 미치는 영향)

  • 이재화;김난선;권태호;박승문;장용석
    • KSBB Journal
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    • v.16 no.4
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    • pp.376-380
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    • 2001
  • The human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was produced from cell suspension culture of transgenic tobacco which was transformed by using Agrobacterium harboring the hGM-CSF gene. To improve the production of hGM-CSF in batch culture system, the effects of initial sucrose concentration and inoculum size were investigated. The results show that the hGM-CSF production was not affected by small inoculum size in medium containing either low or high concentration of sucrose. However, the production of hGM-CSF was increased under increasing of the inoculum sizes and sucrose concentration. Under the combination of inoculum and sucrose concentration, the maximum hGM-CSF production of 720 $\mu$g/L was obtained at 90 g/L of initial sucrose concentration and 110 g/L of inoculum size.

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형질전환된 담배 세포 현탁배양을 이용한 hGM-CSF 생산에서 ultrasound가 미치는 영향

  • Myeong, Hyeon-Jong;Lee, Gi-Yong;Kim, Dong-Il
    • 한국생물공학회:학술대회논문집
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    • 2003.04a
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    • pp.284-287
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    • 2003
  • Effects of ultrasound on cell growth and the production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) were investigated using transgenic Nicotiana tabacum cell suspension cultures. The culture suffered a slight growth depression immediately after the sonication, but gradually recovered to normal growth within 2 days. When the cells were exposed to ultrasound, the level of secreted hGM-CSF was 2.14 times higher than that in normal condition. From the beginning to 6 days of culture, production of secreted hGM-CSF was higher than that of control and then decreased. At the end of culture, however, hGM-CSF was considerably increased up to 36.7%. In the case of intracellular hGM-CSF, the level was slightly higher than that obtained in normal condition. Total hGM-CSF production was 31.5% higher than that of control culture after 6 days. The highest amount of hGM-CSF was $34.9\;{\mu}g/L$.

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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.

The Effects of Light on the Production of hGM-CSF in Transgenic Plant Cell Culture (빛 조사시간에 따른 형질전환된 담배세포 성장과 hGM-CSF의 생산에 미치는 영향)

  • 이재화;이재화;김영숙;홍신영;신윤지;서조은;권태호;양문식
    • KSBB Journal
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    • v.16 no.6
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    • pp.568-572
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    • 2001
  • Light is one of the most important environmental factors controlling plant physiology. The human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was produced from cell suspension cultures of transgenic tobacco under different light conditions (24 hr light, 18 hr light/dark cycle, dark). Under 24 hr light condition, cell growth was best and dry cell weight reached 14.4 g/L. Light did not influenced the secretion of total proteins. However, in the dark condition, the ratio of secreted total protein/dry cell weight was 1.5 fold higher than those of ethel conditions. Production of hGM-CSF was highest with 18 hr light condition and reached 496.5 ug/L. In addition, the content of hGM-CSf in secreted total proteins was 1.8 fold higher than that of 24 hr light condition, which is beneficial for the purificationof the protein.

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Secretory Production of hGM-CSF with a High Specific Biological Activity by Transgenic Plant Cell Suspension Culture

  • Kwon, Tae-Ho;Shin, Young-Mi;Kim, Young-Sook;Jang, Yong-Suk;Yang, Moon-Sik
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.8 no.2
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    • pp.135-141
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    • 2003
  • The human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene was introduced into tobacco plants. The cell suspension culture was established from leaf-derived calli of the transgenic tobacco plants in order to express and secrete a biologically active hGM -CSF. The recombinant hGM-CSF from the transgenic plant cell culture (prhGM-CSF) was identified as a yield of about 180 ${\mu}$g/L in the culture filtrate, as determined by ELISA. The addition of 0.5 g/L polyvinylpyrrolidone (PVP) to the plant cell culture medium both stabilized the secreted prhGM-CSF and increased the level of production approximately 1.5-fold to 270 ${\mu}$g/L. The biological activity of the prhGM-CSF was confirmed by measuring the proliferation of the hGM-CSF-dependent cell line, TF-1. Interestingly, the specific activity of the prhGM-CSF was estimated to be approximately 2.7 times higher than that of a commercially available preparation from E. coli.

Enhanced production of hGM-CSF by temperature shifting in transgenic Nicotiana tabacum cell suspension cultures

  • Kim, Yong-Hoon;Lee, Sang-Yoon;Kim, Dong-Il
    • 한국생물공학회:학술대회논문집
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    • 2003.10a
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    • pp.329-333
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    • 2003
  • Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is a glycoprotein that stimulates the production of granulocytes, macrophages and white blood cells. hGM-CSF secreted by transgenic Nicotiana tabacum suspension cells was unstable in the culture medium and rapidly degraded by extracellular preteases. In order to reduce extracellular pretense activity, culture temperature was lowered. Then, the production of hGM-CSF by transgenic plant suspension cell cultures could be enhanced by reduced degradation of hGM-CSF at low temperature.

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Immobilization of transgenic Nicotiana tabacum cell suspensions for the continuous production of hGM-CSF

  • Roh, Yun-Sook;Lee, Sang-Yoon;Kim, Dong-Il
    • 한국생물공학회:학술대회논문집
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    • 2003.10a
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    • pp.341-345
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    • 2003
  • Effect of immobilization on the production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) by Nicotiana tabacum cells was investigated using polyurethane foam as immobilization matrices. The cell activity and the hGM-CSF production were maintained for 16 days in spite of 3 times of media exchange. Under the same conditions of temperature and agitation rate, maximum concentrations of hGM-CSF in a 500-mL spinner flask and 100-mL Erleuneyer flasks were 17.3 ${\mu}g/L$ and 9.8 ${\mu}g/L$, respectively. Consequently high hGM-CSF production could be possible in spinner flask when the rate and amount of media exchange were optimized.

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Increased production of human granulocyte-macrophage colony stimulating factor (hGM -CSF) by the addition of stabilizing polymer in plant suspension cultures

  • Kim, Nan-Seon;Lee, Jae-Hwa;Kim, Yeong-Suk;Gwon, Tae-Ho;Yang, Mun-Sik
    • 한국생물공학회:학술대회논문집
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    • 2001.11a
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    • pp.95-98
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    • 2001
  • The effect of stabilizing polymer on hGM-CSF production was investigated in suspension cell cultures of transgenic tobacco. Secreted human GM -CSF from cell suspension cultures was detected in the medium at a maximum concentration of 180 ${\mu}g/L$ by ELISA. However, the secreted hGM -CSF was unstable in the medium, and rapidly degraded after day 5. In order to stabilize the secreted hGM-CSF, three stabilizing polymers were tested, polyethylene glycol, polyvinylpyrrolidone and gelatin. Gelatin was the most effective in stabilizing the secreted GM-CSF. Following the addition of 5% (w/v) gelatin, the maximum GM -CSF concentration reached 783 ${\mu}g/L$, a 4.6-fold increase over control.

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Efficient Expression System of High Value Proteins, hGM-CSF and hEGF, using Agrobacterium-mediated Chlorella Transformation (아그로박테리움 매개 클로렐라 형질전환을 이용하여 유용 단백질인 hGM-CSF와 hEGF의 효율적인 발현 시스템)

  • Yu Jeong Jeong;Mi ­ Jung Park;Woo ­ Jeong Lee;Sung Chun Kim
    • Journal of Marine Bioscience and Biotechnology
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    • v.16 no.1
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    • pp.26-35
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    • 2024
  • Chlorella has various biotechnological applications, including in the biomedical and pharmaceutical industries, because of its advantages, including rich nutrients, fast growth rate, easy cultivation, and high biomass. We used the Agrobacterium-mediated transformation method to express human GM-CSF and EGF proteins, which are widely used in regenerative medicine, cosmetics, and pharmaceutical materials in Chlorella. The codon-optimized hGM-CSF and hEGF genes were cloned into plant binary vectors and transformed into Chlorella vulgaris using the Agrobacterium-mediated coculture transformation method. After transformation, genomic DNA PCR was performed for each C. vulgaris line that was stably subcultured on an antibiotic-resistant solid medium to confirm the insertion of hGM-CSF and hEGF into the chromosome. Furthermore, PT-PCR and protein expression of hGM-CSF and hEGF in each transformed C. vulgaris were significantly increased compared to the untransformed Chlorella. This study suggests that high-value proteins, including hGM-CSF and hEGF, which are foreign genes of C. vulgaris, can be stably expressed through the Agrobacterium-mediated Chlorella transformation system.

Optimization of main factors using response surface method for the enhanced production of hGM-CSF from transgenic Nicotiana tabacum cell suspension cultures

  • Lee, Ki-Yong;Lee, Sang-Yoon;Kim, Dong-Il
    • 한국생물공학회:학술대회논문집
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    • 2003.10a
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    • pp.351-355
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    • 2003
  • Response surface methodology was employed to study the interactive effect of sucrose, nitrogen, temperature and to optimize their levels to enhance the production of human granulocyte-macrophage colony-stimulation factor from Nicotiana tabacum cell suspension cultures. A 15-runs Box-Behnken design including three center points was the response surface method selected for the initial set of experiments. The analysis of the data from the Box-Behnken experiments showed interactive effects of sucrose:nitrogen, sucrose:temperature and nitrogen:temperature. The optimal combinations of sucrose, nitrogen and temperature for hGM-CSF production from surface plot were sucrose 90 g/L, nitrogen 41 mM and 22$^{\circ}C$, respectively. The optimization of there factors enhanced the hGM-CSF production by 2 times because high sucrose concentration stimulated the secretion of hGM-CSF and low temperature prevented hGM-CSF degradation in media by pretenses.

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