• Biotechnology and Bioprocess Engineering:BBE
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• v.8 no.3
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• pp.187-191
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• 2003

Proteolytic enzymes existing in plant cell cultured media are the major reason for the loss of secreted human granulocyte-macrophage colony-stimulating factor (hGM-CSF). The addition of pepstatin, aprotinin and PMSF relatively decreased the proteolytic degradation of hGM-CSF in a conditioned medium, but sufficient prevention against the proteolytic activity could not be obtained with chemical protease inhibitors. Gelatin, as a competitive substrate for protease, showed a stabilizing effect in a conditioned medium. Compared to the initial hGM-CSF concentration in a conditioned medium. with 10 g/L of gelatin, 68% of the hGM-CSF remained after 5 days. In a cell culture experiment, 5 g/L of gelatin significantly stimulated the hGM-CSF production and accumulation in culture media, with no growth inhibition. compared to the controls (4.72 $\mu\textrm{g}$/L), the extracellular hGM-CSF level could be increased to 39.78 $\mu\textrm{g}$/L with the addition of 5 g/L of gelatin.

• Journal of Plant Biotechnology
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• v.32 no.4
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• pp.313-319
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• 2005

The effects of various carbon sources on the secretion of hGM-CSF, total protein and protease into the medium were investigated in transgenic tobacco cells. The dry cell weight (11.2 g/L) and wet cell weight (310.8 g/L) were highest at 30 g/L glucose after 5-day culture but, the dry cell weight (13.4 g/L) and wet cell weight (480 g/L) were highest at 30 g/L sucrose after 10-day culture. The total protein (110.3 mg/L), protease activity (3950 U/L) and total secreted hGM-CSF (56 mg/L) were highest at 30 g/L sucrose after 10-day culture. Stabilization of the total secreted protein and hGM-CSF in various carbon source concentrations was determined. Total secreted protein was most stabilized in the medium containing sucrose. However, the loss of the total protein was increased with the concentrations of high level in medium containing sorbitol, mannitol, fructose, and glucose. hGM-CSF was more stabilized in the medium containing sucrose than in the medium containing sorbitol, mannitol, fructose, glucose.

• 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 $34.9\;{\mu}g/L$.

• Journal of Animal Science and Technology
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• v.61 no.2
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• pp.61-68
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• 2019

The hG-CSF (human Granulocyte Colony-Stimulating Factor) is a growth and stimulation factor capable of inducing the proliferation of bone marrow cells, several types of leukocytes, among other hematopoietic tissue cells. hG-CSF is used in used to treat anomalies that reder a small number of circulating white blood cells, which may compromise the immune defenses of the affected person. For these reasons, the production of hG-CSF in a bioreactor system using the mammary gland of genetic modified animals is a possibility of adding value to the bovine genetic material and reducing the costs of hG-CSF production in pharmaceutical industry. In this study, we aimed the production of transgenic hG-CSF bovine through the lipofection of bovine primary fibroblasts with an hG-CSF expression cassette and cloning these fibroblasts by the somatic cell nuclear transfer (SCNT) technique. The bovine fibroblasts transfected with the hG-CSF cassette presented a stable insertion of this construct into their genome and were efficiently synchronized to G0/G1 cell cycle stage. The transgenic fibroblasts were cloned by SCNT and produced 103 transferred embryos and 2 pregnancies, one of which reached 7 months of gestation.

• 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.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.391-392
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• 2000

Tobacco cells transformed with hGM-CSF gene produced $162\;{\mu}g/L$ of hGM-CSF, a valuable therapeutic protein in seven days after inoculation. The protein concentration decreased after the maximum point. It is evidenced that the environment of cell culture was inappropriate for the stability of the protein(e.g., low osmolarity which can cause cell lysis).

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.289-292
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• 2003

Effects of immobilization on the production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) by Nicotiana tabacum cells were investigated using alginate and polyurethane foam as immobilization matrices. Encapsulation of the cells in alginate decreased protein production by 50% compared with that of suspension culture. Maximum hGM-CSF concentration was obtained by the cells immobilizaed in polyurethane foam. High hGM-CSF production could be possible when polyurethane foam was used because of high specific production and easy immobilization for cell recycling process with high cell density.

• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.321-324
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• 2002

In situ recovery of recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) was performed in transgenic Nicotiana tabacum cell suspension cultures. Aqueous two-phase systems (ATPS) were used to utilize its biocompatibility. Transgenic plant cells could be grown up to 15.7 g/L in normal medium and 18.6 g/L in ATPS composed of 6% (w/w) polyethylene glycol (PEG) 20,000 and 10% (w/w) dextran 2,000,000. It was proved that the A TPS was not harmful to cell growth. In addition, It is expected to recover hGM-CSF simultaneously with cell growth. Using this method, maximum hGM-CSF concentration at 1.64 ng/mL was obtained on day 3.

• 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.

• 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.