Enhancement of human granulocyte-colony stimulating factor production in recombinant E. coli using batch cultivation

Development of inexpensive and simple culture media is always favorable for recombinant protein over-expression in E. coli. The effects of medium composition on the production of recombinant human granulocyte-colony stimulating factor (rh-GCSF) were investigated in batch culture of E. coli BL21 (DE3) [pET23a-hgcsf]. First, the optimum medium for production of rh-GCSF was determined; and, then it was shown that mixture of amino acid addition at induction time, which was determined on the basis of amino acids frequency in the recombinant protein, increases recombinant protein expression level significantly. Furthermore, the effect of glucose concentration on productivity of rh-GCSF was investigated; 20 g/l of glucose will result in maximum attainable biomass and rh-GCSF in this process. At optimum conditions, a cell dry weight of 10.5 g/l, an expression level of about 35% of total cellular protein, rh-GCSF concentration of 1.75 ± 0.1 g/l, and overall rh-GCSF yield of 165 ± 5 mg/g were obtained.     

Characterization of recombinant-derived granulocyte-colony stimulating factor (G-CSF).

Human granulocyte colony-stimulating factor (G-CSF), and a mutant having a Ser for Cys substitution at residue 18 were produced in Escherichia coli strain W3110. About 60 mg of pure protein was obtained from 50 g of wet cells with a recovery of about 20%. The proteins were characterized physically and chemically, including determination of disulphide bonds, which were found to exist between residues 37-43 and 65-75. Cys-18 is not involved in disulphide bond formation and was substituted by Ser with no effects on gross protein conformation or biological activity. Both the wild-type and the mutant recombinant-derived proteins, although not glycosylated, possess colony-stimulating activities. In a bioassay using the murine myelomonocytic leukaemic cell line WEH1 3B D+, activities were obtained which were similar to those of natural G-CSF and of a glycosylated recombinant-derived human G-CSF produced in monkey cells.     

High-level secretory production of human granulocyte-colony stimulating factor by fed-batch culture of recombinant Escherichia coli

Secretory production of human granulocyte colony-stimulating factor fusion protein (hG-CSF) by fed-batch culture of Escherichia coli was investigated in both 2.5-L and 30-L fermentors. To develop a fed-batch culture condition that allows efficient production of hG-CSF, different feeding strategies including pH-stat, exponential and constant feeding were examined. Among these, the constant feeding strategy (0.228 g glucose2min^-1) and the exponential feeding that supports a low specific growth rate (µ=0.116 h^-1) resulted in the best hG-CSF production. Under these conditions, 4.4 g2L^-1 of hG-CSF was produced. The effect of induction time on the protein production was also investigated. For the fed-batch cultures carried out with the pH-stat and exponential feeding strategies, induction at higher cell density (late-exponential phase) resulted in more hG-CSF production compared with induction at lower cell density (early to mid-exponential phase). The constant feeding strategy that supported best hG-CSF production was applied to the scale-up production of hG-CSF in 30 L of fermentor. The maximum dry cell weight and hG-CSF concentration of 51.7 and 4.2 g2L^-1, respectively, was obtained.     

Splenomegaly induced by recombinant human granulocyte-colony stimulating factor in rats

Spontaneous ruptures of the spleen have been observed in donors and patients with malignancy during mobilization of peripheral blood stem cells. Thus, we investigated the morphological and histological alteration of the spleen, and mRNA expression levels and activities of the DNA-synthesizing enzymes thymidylate synthase (TS) and thymidine kinase (TK) in the splenic cells, of rats treated with recombinant human granulocyte-colony stimulating factor (rhG-CSF). Daily injections of rhG-CSF for 5 or 7 days slightly enhanced the splenic weight. Single or 3-day treatment with rhG-CSF markedly enhanced the number of granulocytes in peripheral blood. Expression levels of TS and TK mRNA in the splenic cells were significantly increased 6 hours after rhG-CSF treatment. Early expression of TS and TK mRNA in the splenic cells may indicate a reseeding of hematopoietic cells from the bone marrow. Daily injections with rhG-CSF enhanced the TS and TK activities in the splenic cells. As continuous elevations of DNA-synthesizing enzyme activity and spleno-megaly are suggestive of a possible splenic rupture, the monitoring of peripheral granulocytes and splenic size is necessary during the rhG-CSF treatment.     

Lack of interferon-gamma production despite the presence of interleukin-18 during cutaneous wound healing

BACKGROUND: Recently, we have reported a rapid and strong induction of interleukin-18 (IL-18) upon cutaneous injury in mice. In this paper, we investigated a possible role of IL-18 in triggering interferon-gamma (IFN-gamma) production at the wound site. MATERIALS AND METHODS: Expression of IFN-gamma during cutaneous wound healing was analyzed by RNase protection assay, Western blot, ELISA, and immunohistochemical techniques in a murine model of excisional skin repair. RESULTS: We could not detect any IFN-gamma mRNA and protein expression during normal skin repair. Additionally, impaired healing in the genetically diabetic db/db mouse, which was used as a model for a prolonged inflammatory phase of repair, was characterized by largely elevated levels of IL-18 during the late phase of repair and an absence of IFN-gamma. Western blot analysis for T-cell- and monocyte/macrophage-specific marker proteins (CD4, F4/80) clearly revealed the presence of these subsets of leukocytic cells at the wound site, that are known to produce IFN-gamma in response to IL-18. Furthermore, we provide evidence that the presence of transforming growth factor-beta1 (TGF-beta1) at the wound site might reflect a counterregulatory mechanism in IL-18-induced IFN-gamma production, as TGF-beta1 strongly suppressed IL-18/phytohaemagglutinin (PHA)-induced IFN-gamma production by peripheral blood mononuclear cells (PBMC) in vitro. CONCLUSIONS: Normal tissue regeneration processes after cutaneous injury were not dependent on the presence of IFN-gamma in vivo, and IL-18 must serve additional roles rather than inducing IFN-gamma during the healing process.     

Gestational regulation of granulocyte-colony stimulating factor receptor expression in the human placenta

A number of cytokines and their receptors are abundantly expressed at the materno-fetal interface and are thought to have a function in the regulation of placentation. Granulocyte-colony stimulating factor (G-CSF) is expressed by stromal cells in both placental tissue and maternal decidua throughout placentation. In this study, we examined the expression of placental G-CSF receptor (G-CSFR) mRNA and protein throughout gestation by ribonuclease protection assays, Western blotting, and immunohistochemistry. The major placental form of G-CSFR mRNA, corresponding to a membrane-bound form of the protein, was present in first-trimester placental tissues; levels decreased in second- and were highest in third-trimester placental tissues. Two placental G-CSFR molecules, 120 kDa and 150 kDa, were detected in first- and third-, but not second-, trimester tissues. The level of the 150-kDa G-CSFR was greater in the third- than in first-trimester samples. These differences were irrespective of whether or not the patients had received prostaglandin E1 analogues, prostaglandin E1 analogues and oxytocin, oxytocin alone, or mifepristone before labor. We demonstrated by immunohistochemistry that interstitial cytotrophoblast in first- and second-trimester decidual tissue and cytotrophoblast in term fetal membranes express G-CSFR. These data demonstrate that the expression of specific forms of placental G-CSFR is strictly cell type- and developmental stage-specific, and they suggest that G-CSFR may be important in decidual invasion of cytotrophoblast and in trophoblast function during placentation.     

Serum concentrations of granulocyte-colony stimulating factor in complicated Plasmodium falciparum malaria

Involvement of neutrophils in the control of blood parasites in malaria has been reported. Both, mononuclear phagocytes and neutrophils are known to be stimulated by cytokines such as TNF-alpha in order to augment the defence potency against the parasites. Previously, it has been shown that serum-G-CSF concentrations are increased in patients with bacterial sepsis. In vitro studies have shown that P. falciparum - infected erythrocytes induce the release of G-CSF by several cells such as endothelial cells and monocytes, however, nothing is known about G-CSF serum concentrations during the clinical course of severe P. falciparum malaria. Thus, it was the aim of the present study to investigate the time course for G-CSF serum concentrations in patients with complicated P. falciparum malaria, and to correlate these values with other mediators of inflammation and hematopoesis. Twenty-six patients suffering from complicated P. falciparum malaria were included in the study, and 20, age and sex matched, healthy volunteers were used as the negative control group. Serum samples for determination of G-CSF were taken on day 0, 7 and 14, and measured by ELISA. We found significantly increased serum concentrations of G-CSF in patients with complicated P. falciparum malaria on day 0, values decreasing to within the normal range by day 7. A significant correlation was found between G-CSF (d0) and procalcitonin, the parasite count, erythropoietin and macrophage inflammatory protein, however no correlation could be shown for the neutrophil count. In conclusion, on the day of hospital admission, elevated serum concentrations of G-CSF were detected in patients with complicated P. falciparum malaria, which might indicate a role of G-CSF in the acute defence mechanism against the parasites.     

Methanol-induced tertiary and secondary structure changes of granulocyte-colony stimulating factor

We have studied methanol-induced conformational changes in rmethuG-CSF at pH 2.5 by means of circular dichroism (CD), fluorescence and infrared (IR) spectroscopy, and 8-anilino-1-naphthalene sulfonic acid (ANS) binding. Methanol has little effect on the secondary and tertiary structures of rmethuG-CSF when its concentration is in the range of 0 to 20% (v/v). At 30% (v/v) methanol, rmethuG-CSF has ANS binding ability. In the methanol concentration range of 30 to 70% (v/v) the amount of alpha-helix decreases a little, and the tertiary structure decreases significantly. At methanol concentrations above 70% (v/v), a transition to a more helical state occurs, while there is little change in the tertiary structure, and no ANS binding ability. Thermal denaturation studies involving CD have demonstrated that as the methanol concentration increases the melting temperature and the cooperativity of transition decrease, and the transition covers a much wider range of temperature. It seems that the decreased cooperativity means an increase in the concentration of partially folded intermediate states during the unfolding of rmethuG-CSF.     

G-CSF 在缺血性脑卒中动物模型治疗中的研究进展

粒细胞集落刺激因子(G-CSF)是细胞生长因子家族中的一员,主要功能是刺激细胞增殖分化降低细胞凋亡。近年来许多缺血性脑卒中的实验研究显示其能有效改善由于脑卒中等中枢神经系统疾病所致的神经功能缺损,G-CSF用于缺血性脑卒中的治疗具有广阔的前景。文章从G-CSF在缺血性脑卒中的研究进展及其作用机制等方面进行综述。     

Cysteine 17 of recombinant human granulocyte-colony stimulating factor is partially solvent-exposed

Oh-edaet al. have shown instability of granulocyte-colony stimulating factor (G-CSF) upon storage abovepH 7.0 [J. Biol. Chem. (1990)265, 11,432–11,435]. To clarify the mechanism of this instability, the accessibility of a free cysteinyl residue at position 17 for disulfide exchange reaction was examined using a sulfhydryl reagent. The results show that the cysteine is partially solvent-exposed in both glycosylated and nonglycosylated forms, suggesting that the exposure of the cysteine plays a critical role in the instability of the protein. This is supported by the facts that at lowpH where the cysteine is protonated, both proteins have much greater stability and that a Cys17 Ser analog is extremely stable at neutralpH and 37°C. It was observed that the rate of sulfhydryl titration is slower for the glycosylated form than for the nonglycosylated form, suggesting that the cysteine residue is less solvent-exposed for the former protein or that the pK _a is somewhat more basic. In either case, the carbohydrate appears to affect the reactivity of the sulfhydryl group through steric hindrance or alteration in local conformation. Both the glycosylated and nonglycosylated proteins showed essentially identical conformation as determined by circular dichroism, fluorescence, and infrared spectroscopy. Unfolding of these two proteins, induced either by guanidine hydrochloride or bypH, showed an identical course, indicating comparable conformational stability. Contribution of conformational changes to the observed instability at higherpH is unlikely, since little difference in fluorescence spectrum occurs betweenpH 6.0 and 8.0. Based on these observations, G-CSF, whether glycosylated or not, should not be stored above pH 7.0 in solution. On the other hand, G-CSF is extremely stable in acidic solution as expected from the proposed mechanism.     

Radioprotective efficacy of tocopherol succinate is mediated through granulocyte-colony stimulating factor

The purpose of this study was to elucidate the role of granulocyte colony-stimulating factor (G-CSF) induced by α-tocopherol succinate (TS) in protecting mice from total-body irradiation. CD2F1 mice were injected with a radioprotective dose of TS and the levels of cytokine in serum induced by TS were determined by multiplex Luminex. Neutralization of G-CSF was accomplished by administration of a G-CSF antibody and confirmed by cytokine analysis. The role of G-CSF on gastrointestinal tissue protection afforded by TS after irradiation (11 Gy, 0.6 Gy/min of ⁶⁰Co γ-radiation) was determined by analysis of jejunum histopathology for crypt, villi, mitotic figures, apoptosis, and cell proliferation. Our results demonstrate that TS protected mice against high doses of radiation-induced gastrointestinal damage and TS also induced very high levels of G-CSF and keratinocyte-derived chemokine (KC) production in peripheral blood 24 h after subcutaneous administration. When TS-injected mice were administered a neutralizing antibody to G-CSF, there was complete neutralization of G-CSF in circulating blood, and the protective effect of TS was significantly abrogated by G-CSF antibody. Histopathology of jejunum from TS-injected and irradiated mice demonstrated protection of gastrointestinal tissue, yet the protection was abrogated by administration of a G-CSF antibody. In conclusion, our current study suggests that induction of G-CSF resulting from TS administration is responsible for protection from ⁶⁰Co γ-radiation injury.     

Functional role played by the glycosylphosphatidylinositol anchor glycan of CD48 in interleukin-18-induced interferon-gamma production

Interleukin (IL)-18 induces T cells and natural killer cells to produce not only interferon-gamma but also other cytokines by binding to the IL-18 receptor (IL-18R) alpha and beta subunits. However, little is known about how IL-18, IL-18Ralpha, and IL-18Rbeta form a high-affinity complex on the cell surface and transduce the signal. We found that IL-18 and IL-18Ralpha bind to glycosylphosphatidylinositol (GPI) glycan via the third mannose 6-phosphate diester and the second beta-GlcNAc-deleted mannose 6-phosphate of GPI glycan, respectively. To determine which GPI-anchored glycoprotein is involved in the complex of IL-18 and IL-18Ralpha, IL-18Ralpha of IL-18-stimulated KG-1 cells was immunoprecipitated together with CD48 by anti-IL-18Ralpha antibody. More than 90% of CD48 was detected as beta-GlcNAc-deleted GPI-anchored glycoprotein, and soluble recombinant human CD48 without GPI glycan bound to IL-18Ralpha, indicating that CD48 is associated with IL-18Ralpha via both the peptide portion and the GPI glycan. To investigate whether the carbohydrate recognition of IL-18 is involved in physiological activities, KG-1 cells were digested with phosphatidylinositol-specific phospholipase C before IL-18 stimulation. Phosphatidylinositol-specific phospholipase C treatment inhibited the phosphorylation of tyrosine kinases and the following IL-18-dependent interferon-gamma production. These observations suggest that the complex formation of IL-18.IL-18Ralpha. CD48 via both the peptide portion and GPI glycan triggers the binding to IL-18Rbeta, and the IL-18.IL-18Ralpha.CD48.IL-18Rbeta complex induces cellular signaling.     

Generation of transgenic chickens that produce bioactive human granulocyte-colony stimulating factor

We report here the generation of transgenic chickens that produce human granulocyte-colony stimulating factor (hG-CSF) using replication-defective Moloney murine leukemia virus (MoMLV)-based vectors packaged with vesicular stomatitis virus G glycoprotein (VSV-G). The recombinant retrovirus was injected beneath the blastoderm of nonincubated chicken embryos (stage X). Out of 140 injected eggs, 17 chicks hatched after 21 days of incubation and all hatched chicks were found to express vector-encoded hG-GSF gene. The biological activity of the recombinant hG-CSF was significantly higher than its commercially derived E. coli-derived counterpart. Successful germline transmission of the transgene was also confirmed in G(1) transgenic chicks produced from the cross of Go transgenic roosters with nontransgenic hens, but most of the G(1) progeny were dead within 1 month of hatching.     

Effects of ionic strength on the thermal unfolding process of granulocyte-colony stimulating factor

This paper reports the effect of ionic strength on the process of thermal unfolding of recombinant methionyl human granulocyte-colony stimulating factor (rmethuG-CSF) at acid pH. We previously reported that the protein aggregates were formed at the highest temperature at pD 2.1 in the pD range of 5.5-2.1 and that the aggregation proceeded a little at pD 2.1 because of the strong repulsive interaction between the unordered structures that play the role of a precursor for the aggregation. In the present study temperature-dependent IR spectra and far-UV CD spectra were measured for rmethuG-CSF in aqueous solutions containing various concentrations of NaCl at acid pH. Second derivative and curve-fitting analysis were performed to examine the obtained IR spectra. The results revealed that the structure of rmethuG-CSF becomes less stable with increasing ionic strength at all pDs investigated (pD 2.1, 2.5, and 4.0). We have also demonstrated that, at pD 2.1, the temperature at which the protein aggregation starts becomes lower and that the amount of the aggregates becomes larger with the addition of NaCl. This is probably because the addition of NaCl masks the repulsive electrostatic interaction between the unordered structures.     

Inhibition by rapamycin of the lipoteichoic acid-induced granulocyte-colony stimulating factor expression in mouse macrophages

Granulocyte-colony stimulating factor (G-CSF) is a cytokine which involves in anti-inflammation and inflammation as well. Rapamycin is an inhibitor of mTOR which also plays a role in innate immunity. This study investigated the effect of rapamycin on the lipoteichoic acid (LTA)-induced expression of G-CSF in macrophages and its underlying mechanism. Our data show that LTA induced G-CSF expression in RAW264.7 and bone marrow-derived macrophages and that this effect was inhibited by rapamycin. Analysis of the G-CSF 5′ flanking sequence revealed that the −283 to +35 fragment, which contains CSF and octamer elements, was required for maximal promoter activity in response to LTA stimulation. Western blot analyses of proteins that bind to the CSF and octamer element show that LTA increased protein levels of NF-κB, C/EBPβ and Oct-2, and that rapamycin inhibited the LTA-induced increase in Oct-2 protein levels, but not the others. Knockdown of Oct-2 by RNA interference resulted in a decrease in LTA-induced G-CSF mRNA levels. Moreover, forced expression of Oct-2 by transfection with the pCG-Oct-2 plasmid overcame the inhibitory effect of rapamycin on the LTA-induced increase in G-CSF mRNA levels and promoter activity. This study demonstrates that rapamycin reduces G-CSF expression in LTA-treated macrophages by inhibiting Oct-2 expression.     

Recombinant human granulocyte-colony stimulating factor and recombinant human macrophage-colony stimulating factor synergize in vivo to enhance proliferation of granulocyte-macrophage, erythroid, and multipotential progenitor cells in mice

Combinations of low dosages of purified recombinant human (rh) macrophage-colony stimulating factor (M-CSF; also termed CSF-1) and rh granulocyte-colony stimulating factor (G-CSF) were compared alone and in combination for their influence on the cycling rates and numbers of bone marrow and splenic granulocyte-macrophage, erythroid, and multipotential progenitor cells in vivo in mice pretreated with iron-saturated human lactoferrin (LF). LF was used to enhance detection of the stimulating effects of exogenously added CSFs. Concentrations of each CSF that were not active in vivo when given alone were active when given together, with the other CSF. The concentrations of rhM-CSF and rhG-CSF needed to increase progenitor cell cycling in the marrow and spleen were reduced by factors of 40-200 when these CSFs were administered in combination with low dosages of the other CSF. At the concentrations of rhM-CSF and rhG-CSF tested, synergism was not noted on absolute numbers of progenitor cells or total nucleated cell counts per organ or circulating in the blood. These findings may have potential relevance when considered in a clinical setting where the CSFs might be used in combination with other biotherapy and/or chemotherapy.     

Production of bioactive human granulocyte-colony stimulating factor in transgenic rice cell suspension cultures

Human granulocyte-colony stimulating factor (hG-CSF), a human cytokine, was expressed in transgenic rice cell suspension culture. The hG-CSF gene was cloned into the rice expression vector containing the promoter, signal peptide, and terminator derived from a rice alpha-amylase gene Amy3D. Using particle bombardment-mediated transformation, hG-CSF gene was introduced into the calli of rice (Oryza sativa) cultivar Dong-jin. Expression of the hG-CSF gene was confirmed by ELISA and Northern blot analysis. The amount of recombinant hG-CSF accumulated in culture medium from transgenic rice cell suspension culture on the sugar starvation was determined by time series ELISA. Biological activity of the plant derived hG-CSF was confirmed by measuring the proliferation of the AML-193 cells, and was similar to that of the commercial Escherichia coli-derived hG-CSF. In this paper, we discuss the attractive attributes of using rice cell suspension system for the expression of therapeutic recombinant hG-CSF.     

Priming effect of dextran sulphates on lipopolysaccharide-induced tumour necrosis factor production in mice

Abstract Dextran sulphate (DS) 500 (M.W. 500 000) is commonly used as a reticuloendothelial (RE) blocker. We found that lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF) production in sera was enhanced when mice were pretreated with DS500. When mice were pretreated with DS1000 (M.W. 1 000 000), TNF activity in sera was also significantly enhanced by the LPS injection in comparison with the saline-treated group, but not by the pretreatment with the low molecular weight of DS5 (M.W. 5 000), neutral dextran (Dex) 500, or positively-charged diethylaminoethyl dextran (DEAE-Dex) 500. The enhancement of LPS-induced TNF production occurred from 2 h after DS500 pretreatment. Pretreatment with DS500 or DS1000 significantly suppressed the carbon clearance from the blood in mice from 2 h after DS injection, but this suppression was not detected by the pretreatment with DS5, Dex500, or DEAE-Dex500. We suggest that negative-charge and high molecular weight are essential for dextran derivatives to enhance LPS-induced TNF production, and that the enhancing effect of DS is closely related to the suppression of the RE function.     

Aggregation of granulocyte-colony stimulating factor in vitro involves a conformationally altered monomeric state

Aggregation of partially folded intermediates populated during protein folding processes has been described for many proteins. Likewise, partially unfolded chains, generated by perturbation of numerous proteins by heat or chemical denaturants, have also been shown to aggregate readily. However, the process of protein aggregation from native-state conditions is less well understood. Granulocyte-colony stimulating factor (G-CSF), a member of the four-helix bundle class of cytokines, is a therapeutically relevant protein involved in stimulating the growth and maturation of phagocytotic white blood cells. Under native-like conditions (37 degrees C [pH 7.0]), G-CSF shows a significant propensity to aggregate. Our data suggest that under these conditions, native G-CSF exists in equilibrium with an altered conformation, which is highly aggregation prone. This species is enriched in 1-2 M GdmCl, as determined by tryptophan fluorescence and increased aggregation kinetics. In particular, specific changes in Trp58 fluorescence report a local rearrangement in the large loop region between helices A and B. However, circular dichroism, reactivity toward cyanylation, and ANS binding demonstrate that this conformational change is subtle, having no substantial disruption of secondary and tertiary structure, reactivity of the free sulfhydryl at Cys17 or exposure of buried hydrophobic regions. There is no indication that this altered conformation is important to biological activity, making it an attractive target for rational protein stabilization.     

Regulation of lipopolysaccharide-induced tumor necrosis factor production by cyclosporin A in mice primed with muramyl dipeptide

Abstract The effect of cyclosporin A (CsA) on tumor necrosis factor (TNF) or interleukin-6 (IL-6) production was evaluated in vivo in primed or unprimed mice challenged with lipopolysaccharide (LPS). Both pretreatment with BCG infection or with muramyl dipeptide (MDP) prior to LPS challenge resulted in an increase in the cytokine bioactivity level in the blood. CsA administration inhibited the TNF production. In unprimed mice, either normal or sensitized to LPS lethality by galactosamine treatment, a marked decrease in the cytokine level was observed after injection of CsA. After adrenalectomy, the yield of both TNF and IL-6 following LPS injection was markedly elevated but decreased by CsA administration. Ex vivo experiments have shown that the inhibitory effect of CsA could be demonstrated at the level of macrophages from mice previously given the drug. If mice had received MDP, in vitro responses of cells to LPS were enhanced but again CsA decreased the mRNA expression and protein secretion.