Vol 9: Soluble Prokaryotic Overexpression and Purification of Bioactive Human Granulocyte Colony-Stimulating Factor by Maltose Binding Protein and Protein Disulfide Isomerase.Reportar como inadecuado



 Vol 9: Soluble Prokaryotic Overexpression and Purification of Bioactive Human Granulocyte Colony-Stimulating Factor by Maltose Binding Protein and Protein Disulfide Isomerase.


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This article is from PLoS ONE, volume 9.AbstractHuman 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 ba domain of PDI PDIba, 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 PDIba-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 PDIba-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 PDIba-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.



Autor: Do, Bich Hang; Ryu, Han-Bong; Hoang, Phuong; Koo, Bon-Kyung; Choe, Han

Fuente: https://archive.org/







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