• KSBB Journal
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• v.17 no.1
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• pp.33-37
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• 2002

To improve in vitro release kinetic of G-CSF in PLGA microsphere, G-CSF was PEGylated with methoxy polyethylene glycol-aldehyde (mPEG-aldehyde, MW 5000). The majority of G-CSF was mono-PEGylated and it was characterized using SDS-PAGE, HPLC, and peptide mapping. The PLGA microencapsulation with the native, or PEGylated G-CSF was performed using W/O/w method, where the encapsulation efficiency was high. For the high loading of G-CSF to microsphere, G-CSF and PEGylated G-CSF were concentrated and then verified the protein stability using native gel and gel filtration chromatography. In comparison with native G-CSF, PEGylated G-CSF was released during the extended period and its maximum amount of released G-CSF was also increased.

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

• Clinical and Experimental Pediatrics
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• v.46 no.4
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• pp.376-381
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• 2003

Purpose : This study aimed to demonstrate the possible pathogenesis of granulopoiesis in patients of Kawasaki disease(KD) using quantitative analysis of G-CSF, GM-CSF and their CSFr. Methods : The plasma levels of G-CSF, GM-CSF, G-CSFr and GM-CSFr were studied in 14 patients in the acute phase of KD; 13 children with normal peripheral white blood cell counts were used as the normal control group. The plasma concentration of G-CSF, GM-CSF were analyzed by ELISA. The G-CSFr and GM-CSFr on the peripheral granulocytes were analyzed by a quantitative flow cytometric assay and QuantiBRITE, and the quantitative changes of receptors which did not combine with G-CSF and GM-CSF were measured. Results : The total number of leukocytes in KD was similar to normal control group, but the leukocytes increased according to the number of neutrophils. The plasma concentration of G-CSF were decreased similar to normal control group(P=0.133), but that of GM-CSF decreased more than the normal control group(P=0.227). The quantity of G-CSFr, GM-CSFr were revealed to be no less than the normal control(P=0.721, P=0.912). After incubation with excessive G-CSF, the expressed G-CSFr on the neutrophils were decreased in both groups(P=0.554). The quantities of expressions of GM-CSFr on the neutrophil after incubation with the excessive GM-CSF were always increased in both groups(P=0.255). The amount of GM-CSFr of neutrophils are in proportion to total white blood cells (r=0.788, P=0.035), but it wasn't in the case of KD(P=0.644). Conclusion : The leukocytosis in KD that mediated by increasing neutrophil was not correlated with the plasma concentrations of G-CSF and GM-CSF, and the amount of expression of G-CSFr and GM-CSFr on granulocyte. It is possible that the reduction of concentration of GM-CSF results by increasing the active GM-CSFr.

• Journal of Life Science
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• v.21 no.12
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• pp.1778-1783
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• 2011

Hematopoietic cytokines regulate production of blood cells by stimulating proliferation and differentiation of bone marrow cells. Among these hematopoietic cytokines, called hematopoitic growth factors, glranulocyte-colony stimulating Factor (G-CSF), which regulates growth of neutrophils, is one of important therapeutic factors because cancer patients suffer with neutropenia which is severe reduction of neutrophils after chemotherapy. Two groups of recombinant G-CSF have approved and used for therapeutic purposes and many researches are still on-going to produce recombinant G-CSF by different techniques. We engineered human G-CSF with Bombyx specific endoplasmic reticulum (ER) signal sequence, therefore, secretion of human G-CSF protein was improved in Bombyx mori-origined cell line, Bm5. The Bombyx ER signal sequence and human G-CSF matured protein region chimera was further remodeled at the N-terminus of matured G-CSF protein to understand roles of N-terminus on outer cellular secretion and/or production. Three different mutants were generated deleting three amino acids in non alpha-helical region in N-terminus in order to scan important amino acids for G-CSF secretion. One of 3 different N-terminal deletion mutants showed dramatically reduction of secreted amount of G-CSF indicating its important role on secretion. The data suggest that remodeling in non alpha-helical region of N-terminus is also important for recombinant G-CSF production.

• Clinical and Experimental Pediatrics
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• v.46 no.3
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• pp.271-276
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• 2003

Purpose : Granulocyte-colony stimulating factor(G-CSF) and granulocyte macrophage-colony stimulating factor(GM-CSF) are principal cytokines in granulopoiesis and their physiologic effects are mediated through binding to specific cell surface receptors. Although it is known that the level of serum G-CSF and GM-CSF, and presentation of the receptors are increased in infectious diseases, there have been no studies to find the correlation between the granulopoiesis and leukocytosis. This study was designed to measure G-CSF and GM-CSF in leukocytosis and in control and to demonstrate the possible pathogenesis of granulopoiesis in leukocytosis using quantitative analysis of G-CSF, GM-CSF and their CSFr. Methods : The plasma levels of G-CSF, GM-CSF of 13 children without leukocytosis and 14 children with leukocytosis were measured. Counts of cell surface G-CSFr and GM-CSFr were measured by combining anti G-CSFr and anti GM-CSFr monoclonal antibodies to their respective receptors by using quantitative flow cytometric assay. Results : There was no significant difference betweeen the plasma concentration of G-CSF and GM-CSF in acute leukocytosis and in the control group. However, levels of G-CSFr in acute leukocytosis decreased significantly compared to the control(P=0.012) and the levels of GM-CSFr in both groups revealed no significant difference. Conclusion : Increase in the number of leukocyte in leukocytosis was mediated by increasing the number of neutrophil, and increased plasma concentration of G-CSF may be the cause of neutrophilia. But GM-CSF did not have any influence on leukocytosis.

• Development and Reproduction
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• v.13 no.3
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• pp.191-198
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• 2009

In an attempt to simultaneously produce two human proteins, hGH and hG-CSF, in the milk of transgenic mice, we constructed goat $\beta$-casein-directed hGH and hG-CSF expression cassettes individually and generated transgenic mice by co-injecting them into mouse zygotes. Out of 33 transgenic mice, 29 were identified as double transgenic harboring both transgenes on their genome. All analyzed double transgenic females secreted both hGH and hG-CSF in their milks. Concentrations ranged from 2.1 to $12.4\;mg/m{\ell}$ for hGH and from 0.04 to $0.13\;mg/m{\ell}$ for hG-CSF. hG-CSF level was much lower than hGH level but very similar to that of single hG-CSF mice, which were introduced with hG-CSF cassette alone. In order to address the causes of concentration difference between hGH and hG-CSF in milk, we examined mRNA level of hGH and hG-CSF in the mammary glands of double transgenic mice and tissue specificity of hG-CSF mRNA expression in both double and single transgenic mice. Likewise protein levels in milk, hGH mRNA level was much higher than hG-CSF mRNA, and hG-CSF mRNA expression was definitely specific to the mammary glands of both double and single transgenic mice. These results demonstrated that two transgenes have distinct transcriptional potentials without interaction each other in double transgenic mice although two transgenes co-integrated into same genomic sites and their expressions were directed by the same goat $\beta$-casein promoter. Therefore goat $\beta$-casein promoter is very useful for the multiple production of human proteins in the milk of transgenic animals.

• Journal of Life Science
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• v.21 no.11
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• pp.1652-1657
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• 2011

The production of blood cells is regulated by more than 20 different growth factors, called hematopoitic growth factors. These factors have been produced in prokaryotic and mammalian systems for their clinical use. Glranulocyte-Colony Stimulating Factor (G-CSF) is an important therapeutic factor for cancer patients as well as patients with congenital conditions. These patients do not have enough neutrophils and have a high risk of infection. Two groups of recombinant G-CSF have been used to specially treat cancer patients after chemotherapy because chemotherapy induces neutropenia, a major side effect of chemotherapy drugs. Here, structural and biological characteristics of G-CSF are presented. In addition, the relationship between chemotherapy and neutropenia, which is a severe reduction of neutrophils in the blood, and clinical application of G-CSF is discussed. Recombinant G-CSFs are grouped in two forms. Non-glycosylated G-CSF, filgrastim, is produced in Escherichia coli and glycosylated G-CSF, lenograstim, is produced in Chinese hamster ovary cells. Differences in structure and biological activity are compared and challenges for biosimilar production are also highlighted.

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

• 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.

• Clinical and Experimental Pediatrics
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• v.45 no.4
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• pp.439-448
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• 2002

Purpose : The aim of this study is to determine and compare the effects of adjunctive therapy with different doses of recombinant human granulocyte-colony stimulating factor(rhG-CSF) on reversing sepsis-associated neonatal neutropenia, and their survival rate in a group I/II-type trial. Methods : RhG-CSF was injected subcutaneously to 10 septic-neutropenic neonates with doses of $10{\mu}g/kg$ from Oct. 1995 to Sep. 1996, and was administered to another 12 septic-neutropenic neonates with doses of $5{\mu}g/kg$ from Oct. 1996 to Sep. 1997. Neutrophilic responses and the outcomes of both groups were compared. Results : In the rhG-CSF $10{\mu}g/kg$ treated group and in the $5{\mu}g/kg$ treated group, the absolute neutrophil count(ANC) was $1,065{\pm}89$($mean{\pm}SEM$) and $1,053{\pm}131$, respectively. The only difference between the two groups was the peak ANC at 48 hours. Eight patients from the remaining nine of rhG-CSF $10{\mu}g/kg$ treated group(88.9％) and ten in $5{\mu}g/kg$ treated group(83.3％) survived the sepsis and were discharged without any problems. Conclusions : RhG-CSF can increase the neutrophil count in critically ill septic neutropenic neonats. The survival rate of both groups were up to 90％. This finding suggests that both doses of rhG-CSF may be effective in a therapeutically useful time frame to treat septic neonates with neonatal neutropenia attributable to bone marrow supression or neutrophil consumption.