• Proceedings of the Korean Society of Developmental Biology Conference
• /
• 2005.10a
• /
• pp.130-130
• /
• 2005

• Proceedings of the Korean Society of Embryo Transfer Conference
• /
• 2005.10a
• /
• pp.130-130
• /
• 2005

• 한국생물공학회:학술대회논문집
• /
• 2001.11a
• /
• pp.447-448
• /
• 2001

Human garnulocyte-colony stimulating factor(hG-CSF), a hematopoietic growth factor$^{1)}$, was produced and secreted from tobacco cell suspension. hG-CSF produced from tobacco cell suspension culture is biologically active form. The produced amount of hG-CSF is about 100${\mu}g/L$ in 9 days after inoculation.

• Biomolecules & Therapeutics
• /
• v.6 no.4
• /
• pp.400-405
• /
• 1998

The pharmacokinetics of CJ-50001 (recombinant human granulocyte-colony stimulating factor, developed by R&D center of Cheil Jedang Corp.) were investigated in rats and dogs. The serum concentrations of CJ-50001 were measured by a sandwich enzyme immunoassay. After single intravenous (iv) administration of Cf-50001 to rats at a dose of 5 $\mu$g/kg, the mean terminal half-life and area under the concentration-time curve (AUC) were 0.96 h and 124.497g . h/ml, respectively. After single subcutaneous (sc) administration at the same dose, maximum serum concentration was observed at about 2 hours after administration, and the mean terminal half-life, AUC and the bioavailability were 1.11 h,63.58$\mu$g . h/ml and 51.07%, respectively. In repeated dosing studies, CJ-50001 was administered iv and sc to rats at a daily dose of 5$\mu$g/kg for 7 days. The pharmacokinetic parameters, such as mean AUC and terminal half-life, were no significantly different from those of single administration. Following single iv and sc administration of CJ-50001 to dogs at a dose of 5 $\mu$g/kg, mean AUCs were much higher than those of rats, due to the decreased clearence (CL). After sc administration to dogs, maximum serum concentration was observed at 2~4 hours after administration and the bioavailability was 54.60%.

• Journal of Life Science
• /
• v.20 no.11
• /
• pp.1577-1581
• /
• 2010

Granulocyte colony stimulating factor (G-CSF) is a hematopoietic cytokine that stimulates bone marrow cells to proliferate and differentiate into granulocytes. G-CSF is approved and used for therapeutic purposes. The endoplasmic reticulum (ER) signal peptide of hG-CSF was replaced with silkworm-specific signal peptides to express and efficiently secrete recombinant hG-CSF by silkworm cells. Plasmids that contain cDNAs for hG-CSF and hG-CSF fused with silkworm- specific signal peptides of prophenoloxidase activating enzyme (PPAE), protein disulfide isomerase (PDI), and bombyxin (BX) were constructed. The G-CSF protein was expressed in insect cell line BM5 and was detected by western blot analysis. The cells transfected with plasmids containing rhG-CSF genes with silkworm-specific signal sequences released mature rhG-CSF protein more efficiently than the cells transfected with pG-CSF, the plasmid containing human G-CSF gene, including its own signal sequence. The production of hG-CSF reached maximal level at four days post-transfection and remained at a high level until 7 days post-transfection. These data demonstrate that the modification of the human G-CSF mimic to insect proteins synthesized in ER greatly improves the production of the protein.

• Journal of Microbiology and Biotechnology
• /
• v.10 no.3
• /
• pp.321-326
• /
• 2000

To examine the feasibility of the long-term production of the human granulocyte colony stimulating factor (hG-CSF) using the $_{L}-arabinose$ promoter system of Escherichia coli, flask relay culture and cyclic fed-batch culture were performed. In the flask relay culture, it was found that the pismid was maintained stably up to about 170 generations in an uninduced condition, whereby the cells could also maintain the capability of expressing hG-CSF expression were maintained stably up to at least 100 generations. In contrast, in the cyclid fed-batch culture, segregational plasmid instability was observed within about 4 generations after induction, even though the cell growth and hG-CSF production reached their maximum balues, 78.0 g/l of dry cell weight and 7.0 g/l of hG-CSF, respectively. It would appear that, when compared to the flask relay culture, the high-cell density and high-level expression of hG-CSF in the cyclic fed-batch cultrure led to the segregational plasmid instability; in other words, a severe metabolic burden existe on the cells due to the high-level expression of hG-CSF. Accordingly, based on these long-term cultures, the segregational and structural plasmid instability was observed and a strategy to overcome such problems could be designed.

• Journal of Microbiology and Biotechnology
• /
• v.15 no.6
• /
• pp.1267-1272
• /
• 2005

The effects of coexpression of GroEL/ES and DnaK/DnaJ/GrpE chaperones on the productivity of the soluble form of human granulocyte colony stimulating factor (hG-CSF) in E. coli were examined. Recombinant hG-CSF protein was coexpressed with DnaK/DnaJ/GrpE or GroEL/ES chaperones under the control of the araB or Pzt-1 promoter, respectively. The optimal concentration of L-arabinose for the expression of DnaK/DnaJ/GrpE was found to be 1 mg/ml. When L-arabinose was added at $OD_{600}$=0.2 (early-exponential phase), soluble hG-CSF production was greatly increased. In addition, it was observed that the DnaK/DnaJ/GrpE and GroEL/ES chaperones had no synergistic effects on preventing aggregation of hG-CSF protein. Consequently, by coexpression of the DnaK/DnaJ/GrpE chaperone, the signal intensity of the hG-CSF protein band in the soluble fraction of cell lysate was increased from $3.5\%\;to\;13.9\%$, and Western blot analysis also revealed about a 4-5-fold increase of production of soluble hG-CSF over the non-induction case of DnaK/DnaJ/GrpE.

• Microbiology and Biotechnology Letters
• /
• v.32 no.2
• /
• pp.135-141
• /
• 2004

Human granulocyte colony-stimulating factor (hG-CSF) is a hematopoiesis agent that principally affects the differentiation of neutrophils in the bone marrow. At present, recombinant hG-CSF is used successfully in the treatment of chemotheraphy-induced neutropenia and its indication has been expanded to bone marrow transplantation and aplastic anemia. In this study, we have constructed rhG-CSF secretion plasmid pYRC1 in which OmpA signal sequence/hG-CSF gene was expressed under the control of the T7 promoter. rhG-CSF produced in E. coli BL21 (pYRC1) grown at $37{\circ}C$ was found in aggregates. However, 15% of the periplasmic protein was soluble rhG-CSF when the E. coli BL21 (pYRC1) was cultured at $25^{\circ}C$ for 7 h in the modified MBL medium containing 10 g/$\ell$ glucose with 10 $\mu$M IPTG induction. The production of soluble rhG-CSF in E. coli BL21 (pYRC1) using fed batch culture was also studied. In the fed batch culture system, the final yield of rhG-CSF produced from E. coli BL21 (pYRC1) was increased from 4.4 mg/$\ell$to 24 mg/$\ell$by controlling the specific growth rate from $0.43 h^{-1}$ to $0.14 h^{-1}$, and optimizing the time of induction.

• 한국생물공학회:학술대회논문집
• /
• 2002.04a
• /
• pp.325-328
• /
• 2002

Production of human granulocyte-macrophage colony stimulating factor (hGM-CSF) by Nicotiana tabacum cell suspension culture was studied in Murashige and Skoog (MS) medium with sucrose as a carbon source, ammonium nitrate and potassium nitrate as nitrogen sources, potassium dihydrogen phosphate and sodium dihydrogen phosphate hydrate as phosphate sources, respectively. Optimum concentrations for carbon, nitrogen, phosphate was determined to enhance the production of hGM-CSF. Cell growth was better at high initial sucrose concentration (60 g/L), high initial nitrogen concentration (121.04 mM). Maximum cell density (18.28 g/L) was obtained at 60 g/L of sucrose after 14 days. Cell growth was not so good at low initial sucrose concentration 00 g/L), but the highest hGM-CSF production was obtained at the latter half of exponential phase. hGM-CSF production increased about 3 fold at initial phosphate concentration of 4.96 nM

• Journal of Life Science
• /
• v.18 no.7
• /
• pp.912-917
• /
• 2008

To investigate the biological activity of recombinant human granulocyte colony-stimulating factor (rec-hG-CSF) in mammalian cells, hG-CSF gene was cloned using the eDNA extracted from the human squamous carcinoma cell lines and rec-hG-CSF was produced in CHO cell lines. To analyze the biological activity in vivo, the rec-hG-CSF protein was injected into mice subcutaneously on days 0 and 2. Blood was withdrawn for white blood cell (WBC) determination 5 days after the first injection. WBC values were found to have increased significantly. A pEGFP-mUII-hG-CSF vector was transfected into somatic cell lines isolated from bovine fetal cells. The colony expressing EGFP signals was observed with a confocal microscope. These data suggest that the rec-hG-CSF produced in this study has potent activity in vivo. Thus, the results of this biological activity show that rec-hG-CSF can be enhanced considerably by genetic engineering that affects potential activity, including mutations, which add the oligosaccharide chain and construct double-fusion proteins. A pEGFP-mUII-hG-CSF vector can be utilized for the production of cloned transgenic livestock.