Enhanced biological effects of Phe140Asn, a novel human granulocyte colony-stimulating factor mutant, on HL60 cells

Chung, Hee Kyoung; Kim, Sung Woo; Byun, Sung June; Ko, Eunyoung; Chung, Hak-Jae; Woo, Jae-Seok; Yoo, Jae Gyu; Lee, Hwi-Cheul; Yang, Byoung‐Chul; Kwon, Moosik; Park, Soo-Bong; Park, Jin-Ki; Kim, Kyung-Woon

doi:10.5483/bmbrep.2011.44.10.686

Cited by 8 publications

(7 citation statements)

References 25 publications

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“…As a result of the non-optimal spatial orientation of the molecules, the activity of the GCSF dimer is much lower than that of the GCSF monomer in vitro [62]. Some effective solutions, such as the mutation of Cys 18 [21], [36] or the addition of a specific secretory signal peptide that directs the secretion of hGCSF into the periplasmic space [24], have been used to overcome this obstacle in E. coli . Here, soluble monomeric hGCSF with bioactivity similar to that of hGCSF purified from HEK cells was obtained using a fusion protein strategy and a low expression temperature.…”

Section: Discussionmentioning

confidence: 99%

“…The M-NFS-60 mouse myelogenous leukemia cell line [34], [35], kindly provided by Dr. Kyung-Woon Kim (Rural Development Administration, Suwon, Korea), was grown in RPMI-1640 medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum, 1X penicillin and streptomycin (Invitrogen), and 0.05 mM β-mercaptoethanol [36]. The cells were maintained at 37°C in a humidified atmosphere containing 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

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Soluble Prokaryotic Overexpression and Purification of Bioactive Human Granulocyte Colony-Stimulating Factor by Maltose Binding Protein and Protein Disulfide Isomerase

et al. 2014

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Human granulocyte colony-stimulating factor (hGCSF), a neutrophil-promoting cytokine, is an effective therapeutic agent for neutropenia patients who have undergone several cancer treatments. Efficient production of hGCSF using E. coli is challenging because the hormone tends to aggregate and forms inclusion bodies. This study examined the ability of seven different N-terminal fusion tags to increase expression of soluble hGCSF in E. coli. Four tag proteins, namely maltose-binding protein (MBP), N-utilization substance protein A, protein disulfide isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), increased the solubility of hGCSF under normal conditions. Lowering the expression temperature from 30°C to 18°C also increased the solubility of thioredoxin-tagged and glutathione S-transferase-tagged hGCSF. By contrast, hexahistidine-tagged hGCSF was insoluble at both temperatures. Simple conventional chromatographic methods were used to purify hGCSF from the overexpressed PDIb'a'-hGCSF and MBP-hGCSF proteins. In total, 11.3 mg or 10.2 mg of pure hGCSF were obtained from 500 mL cultures of E. coli expressing PDIb'a'-hGCSF or MBP-hGCSF, respectively. SDS-PAGE analysis and silver staining confirmed high purity of the isolated hGCSF proteins, and the endotoxin levels were less than 0.05 EU/µg of protein. Subsequently, the bioactivity of the purified hGCSF proteins similar to that of the commercially available hGCSF was confirmed using the mouse M-NFS-60 myelogenous leukemia cell line. The EC50s of the cell proliferation dose-response curves for hGCSF proteins purified from MBP-hGCSF and PDIb'a'-hGCSF were 2.83±0.31 pM, and 3.38±0.41 pM, respectively. In summary, this study describes an efficient method for the soluble overexpression and purification of bioactive hGCSF in E. coli.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Soluble Prokaryotic Overexpression and Purification of Bioactive Human Granulocyte Colony-Stimulating Factor by Maltose Binding Protein and Protein Disulfide Isomerase

et al. 2014

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“…G-CSF stimulates the survival, proliferation, differentiation and function of neutrophilic precursor (32,33). M-CSF and GM-CSF are known to be factors of the lungs which are essential in the differentiation of myeloid cells and the immune response (34,35).…”

Section: Resultsmentioning

confidence: 99%

Mycobacterium tuberculosis-induced expression of granulocyte-macrophage colony stimulating factor is mediated by PI3-K/MEK1/p38 MAPK signaling pathway

Cho

Park

Lee³

et al. 2013

BMB Reports

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Members of the colony stimulating factor cytokine family play important roles in macrophage activation and recruitment to inflammatory lesions. Among them, granulocyte-macrophage colony stimulating factor (GM-CSF) is known to be associated with immune response to mycobacterial infection. However, the mechanism through which Mycobacterium tuberculosis (MTB) affects the expression of GM-CSF is poorly understood. Using PMA-differentiated THP-1 cells, we found that MTB infection increased GM-CSF mRNA expression in a dosedependent manner. Induction of GM-CSF mRNA expression peaked 6 h after infection, declining gradually thereafter and returning to its basal levels at 72 h. Secretion of GM-CSF protein was also elevated by MTB infection. The increase in mRNA expression and protein secretion of GM-CSF caused by MTB was inhibited in cells treated with inhibitors of p38 MAPK, mitogen-activated protein kinase kinase (MEK-1), and PI3-K. These results suggest that up-regulation of GM-CSF by MTB is mediated via the PI3-K/MEK1/p38 MAPK-associated signaling pathway. [BMB Reports 2013; 46(4): 213-218]

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“…The carbohydrate chain of the glycoprotein is critical for its biological activities such as stability, half-life, and solubility (12,13). In our previous report, a novel N -linked hG-CSF (Phe140Asn) had enhanced biological activity in HL60 cells (14). Herein, we investigate another function of this mutant glycosylation in heart failure.…”

Section: Introductionmentioning

confidence: 88%

Antiapoptotic effects of Phe140Asn, a novel human granulocyte colony-stimulating factor mutant in H9c2 rat cardiomyocytes

Chung¹,

Ko²,

Kim³

et al. 2012

BMB Reports

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Granulocyte colony-stimulating factor (G-CSF) is used for heart failure therapy and promotes myocardial regeneration by inducing mobilization of bone marrow stem cells to the injured heart after myocardial infarction; however, this treatment has one weakness in that its biological effect is transient. In our previous report, we generated 5 mutants harboring N-linked glycosylation to improve its antiapoptotic activities. Among them, one mutant (Phe140Asn) had higher cell viability than wild-type hG-CSF in rat cardiomyocytes, even after treatment with an apoptotic agent (H2O2). Cells treated with this mutant significantly upregulated the antiapoptotic proteins, and experienced reductions in caspase 3 activity and PARP cleavage. Moreover, the total number of apoptotic cells was dramatically lower in cultures treated with mutant hG-CSF. Taken together, these results suggest that the addition of an N-linked glycosylation was successful in improving the antiapoptotic activity of hG-CSF, and that this mutated product will be a feasible therapy for patients who have experienced heart failure. [BMB Reports 2012; 45(12): 742-747]

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Enhanced biological effects of Phe140Asn, a novel human granulocyte colony-stimulating factor mutant, on HL60 cells

Cited by 8 publications

References 25 publications

Soluble Prokaryotic Overexpression and Purification of Bioactive Human Granulocyte Colony-Stimulating Factor by Maltose Binding Protein and Protein Disulfide Isomerase

Soluble Prokaryotic Overexpression and Purification of Bioactive Human Granulocyte Colony-Stimulating Factor by Maltose Binding Protein and Protein Disulfide Isomerase

Mycobacterium tuberculosis-induced expression of granulocyte-macrophage colony stimulating factor is mediated by PI3-K/MEK1/p38 MAPK signaling pathway

Antiapoptotic effects of Phe140Asn, a novel human granulocyte colony-stimulating factor mutant in H9c2 rat cardiomyocytes

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