Soluble periplasmic production of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens

Jin, H; Cantin, Greg T.; Maki, Steven L.; Chew, Lawrence; Resnick, Sol M.; Ngai, Jerry; Retallack, Diane M.

doi:10.1016/j.pep.2011.03.002

Cited by 22 publications

(12 citation statements)

References 21 publications

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“…The EC 50 values for hGCSF purified from MBP-hGCSF (2.83 pM) and PDIb'a'-hGCSF (3.38 pM) were consistent with a previous study that reported an EC 50 value in the range of 0.8–6 pM for hGCSF [25], [54], [55]. At high concentrations, the purified hGCSF proteins induced mild inhibition of cell proliferation, resulting in a bell-shaped biphasic dose-response curve (Figure 5E).…”

Section: Discussionsupporting

confidence: 91%

“…Escherichia coli also produces aggregated hGCSF in inclusion bodies (IBs) [16]–[22]; however, the overall yield of biologically active protein from these structures is usually low [23]. Alternatively, hGCSF can be secreted into the periplasm of E. coli [24], [25], although low yields are also usually obtained using this method. Maltose-binding protein (MBP), and stress-responsive proteins such as peptidyl-prolyl cis-trans isomerase B, bacterioferritin, and glutathione synthase, have previously been tested as fusion partners to increase the production of solubilized hGCSF in E. coli [26], [27].…”

Section: Introductionmentioning

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: Discussionsupporting

confidence: 91%

Section: Introductionmentioning

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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“…Those methods are widely applied for enzymes, hormones, and growth factors: For the granulocyte colony-stimulating factor (G-CSF), the NFS-60 proliferation assay is used (Apte-Deshpande et al, 2009;Jin et al, 2011). For enzymes, a selective metabolization of a substrate or the accumulation of a product is evaluated (Forootanfar et al, 2011;Su and Chiang, 2006).…”

Section: Introductionmentioning

confidence: 99%

Integrated development of up- and downstream processes supported by the Cherry-Tag™ for real-time tracking of stability and solubility of proteins

Baumann

Bluthardt

Renner

et al. 2015

Journal of Biotechnology

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“…Some biopharmaceuticals require post‐translational modifications, as for example, for complete proteins' glycosylation or suitable production of SS‐rich proteins, so that the absence of such abilities in E. coli represents a significant limitation for synthesis of biomolecules . Among prokaryotic hosts, Bacillus spp, Staphylococcus carnosus , Pseudomonas fluorescens and Ralstonia eutropha have already been studied. Although these strains present fewer disadvantages compared with E. coli, their use for the production of heterologous protein is not well established and several efforts have been performed to improve their production process.…”

Section: Introductionmentioning

confidence: 99%

Liquid–liquid extraction of biopharmaceuticals from fermented broth: trends and future prospects

Santos

Santos-Ebinuma

Pessoa

et al. 2017

J of Chemical Tech & Biotech

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Biopharmaceuticals are one of the most important groups of biotechnological products, representing one-quarter of all pharmaceutical sales and providing suitable and efficient medical care for many previously untreatable diseases. However, their production and purification usually require complex processes, resulting in extremely expensive final products. Thus, the development of newer, simpler and cheaper methods for biosynthesis and purification of these biocompounds, as well as proper integration between these stages, are crucial to reduce their commercial costs and to increase the scale of biopharmaceuticals' medical use. One solution for this concern relies on the proper integration between upstream and downstream processing applying liquid-liquid extraction systems as an intermediate stage. Recently, several works reported that a proper choice of liquid-liquid systems such as aqueous biphasic systems (ABS) can be a suitable platform to simplify, reduce the number of the downstream stages, improve the recovery and purification yields and, consequently, decrease biopharmaceuticals' manufacturing costs. This review will explain the general concept of biopharmaceuticals, their main production and purification methods, particularly, exploring the use of ABS as effective and integrative platforms for their recovery and purification, and providing further insights into the future trends and prospects in the field.

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Soluble periplasmic production of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens

Cited by 22 publications

References 21 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

Integrated development of up- and downstream processes supported by the Cherry-Tag™ for real-time tracking of stability and solubility of proteins

Liquid–liquid extraction of biopharmaceuticals from fermented broth: trends and future prospects

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