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

Wingfield, Paul T.; Benedict, Robert C.; Turcatti, Gerardo; Allet, Bernard; Mermod, Jean‐Jacques; DeLamarter, J F; Simona, M G; Rose, Keith

doi:10.1042/bj2560213

Cited by 26 publications

(14 citation statements)

References 37 publications

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“…A previous study in which Cys 18 was mutated to Ser demonstrated that Cys 18 is not required for bioactivity of hGCSF [60]. However, during folding of hGCSF, intermolecular disulfide bonds between two Cys 18 residues or Cys 18 and another Cys residue can occur in aggregates [61].…”

Section: Discussionmentioning

confidence: 98%

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: 98%

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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“…This protein has five Cys residues that form two disulphide bonds between Cys37-Cys43 and Cys65-75, and another Cys18 a free Cys residue which is not involved in any disulphide bond formation (Wingfield, 1988). It is complicated to recover soluble protein from IBs related to initial recovery, solubilization, and renaturation steps, hence, fusion partners are commonly considered to obtain the soluble protein (Thatcher, 1990).…”

Section: Resultsmentioning

confidence: 99%

“…Post translational modification is not an issue for hG-CSF because O-glycosilated bond does not bind with receptor, so both of the wild-type and non-glycosylated mutant recombinant hG-CSF possess colony stimulating activities (Wingfield, 1988;Hill, 1993). However, the Oglycosilated bond has an important role to avoid aggregation, thus, hG-CSF is frequently produced in E. coli as inclusion bodies (Yamamoto, 2002;Dasari, 2008).…”

Section: Introductionmentioning

confidence: 99%

SOLUBLE EXPRESSION OF SYNTHETIC CSF3syn GENE FUSED WITH THIOREDOXIN IN Escherichia coli BL21(DE3) THROUGH AUTOINDUCTION METHOD AND PURIFICATION

Pratiwi

Fuad

2015

Indonesian J. Pharm.

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A synthetic human gene of CSF3 (CSF3syn.Ec3), coding for hG-CSF was succesfully subcloned into pET32a(+) expression vector and fused with thioredoxin (Trx) at its N-terminal as fusion partner. The obtained fusion gene of Trx-CSF3syn within the recombinant plasmid pET32a(+)_CSF3yn.Ec3 was verified by PCR, plasmid restriction, and DNA sequencing analysis. In order to investigate the fusion gene expression, we transformed Escherichia coli BL21(DE3) as the host with the recombinant plasmid. The gene was succesfully expressed within the cytosol as fusion protein of Trx·tag, His·tag, S·tag, EK-site, and hG-CSF moieties. By the auto induction method, 49% of the protein was found in the soluble fraction and the other 51% was found in the insoluble fraction. The soluble fraction was subsequently purified by IMAC method (Ni-NTA) and characterized.

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“…Kuga et al [7] described a procedure involving IB solubilization, refolding by dialysis and DEAE-Sepharose chromatography. A purification process based on size-exclusion chromatography (Sephacryl S-200), renaturation and CMSepharose with overall recovery of about 20% was reported by Wingfield et al [8].…”

Section: Introductionmentioning

confidence: 94%

“…Several methods based on multiple chromatographic steps have been reported for the purification of rhG-CSF [7][8][9].…”

Section: Introductionmentioning

confidence: 99%

Process development for production of human granulocyte-colony stimulating factor by high cell density cultivation of recombinant Escherichia coli

Khalilzadeh

Mohammadian-Mosaabadi

Bahrami

et al. 2008

J Ind Microbiol Biotechnol

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The fed-batch process using glucose as the sole source of carbon and energy with exponential feeding rate was carried out for high cell density cultivation of recombinant Escherichia coli BL21 (DE3) expressing human granulocyte-colony stimulating factor (hG-CSF). IPTG was used to induce the expression of hG-CSF at 48 g dry cell wt l(-1) during high cell density culture of recombinant E. coli BL21 (DE3) [pET23a-g-csf]. The final cell density, specific yield and overall productivity of hG-CSF were obtained as approximately 64 g dry cell wt l(-1), 223 mg hG-CSF g(-1) dry cell wt and 775 mg hG-CSF l(-1) h(-1), respectively. The resulting purification process used cell lysis, inclusion body (IB) preparation, refolding, DEAE and Butyl-Sepharose. Effects of different process conditions such as cell lysis and washing of IB were evaluated. The results reveal that the cells lyzed at 1,200 bar, 99.9% and Triton removed about 64% of the LPS but sarcosyl had no effect on removal of nucleic acids and LPS. Further analysis show that DEAE column removes DNA about 84%. Cupper concentration was identified as parameter that could have a significant impact on aggregation, as an unacceptable pharmaceutical form that decrease process yields. The purity of purified hG-CSF was more than 99%. Also the comparison of activity between purified hG-CSF and commercial form do not show valuable decrease in activity in purified form.

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

Cited by 26 publications

References 37 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

SOLUBLE EXPRESSION OF SYNTHETIC CSF3syn GENE FUSED WITH THIOREDOXIN IN Escherichia coli BL21(DE3) THROUGH AUTOINDUCTION METHOD AND PURIFICATION

Process development for production of human granulocyte-colony stimulating factor by high cell density cultivation of recombinant Escherichia coli

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