Abstract
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.
Highlights
Granulocyte colony-stimulating factor (GCSF), known as pluripoietin, controls the production, differentiation, and function of granulocytes, which account for 70% of white blood cells [1,2]
Coli, the following seven tags were fused to the N-terminus of the protein via LR recombination cloning: His6, Trx, glutathione S-transferase (GST), protein disulfide isomerase (PDI) b’a’, Maltose-binding protein (MBP), PDI, and Nutilization substance protein A (NusA) (Figure 1)
A TEVrs was inserted between each tag and Human GCSF (hGCSF) to facilitate removal of the tags during purification, and the sequence was codon-optimized for E. coli expression (Figure 1B)
Summary
Granulocyte colony-stimulating factor (GCSF), known as pluripoietin, controls the production, differentiation, and function of granulocytes, which account for 70% of white blood cells [1,2]. Production of GCSF, which is secreted predominantly by macrophages, fibroblasts and endothelial cells, is stimulated by several inflammatory stimuli, including interleukin-1b, tumor necrosis factor-alpha, and lipopolysaccharide [6,7,8]. Human GCSF was initially purified from a tumor cell line that continuously secreted the protein [14]. Escherichia coli produces aggregated hGCSF in inclusion bodies (IBs) [16,17,18,19,20,21,22]; the overall yield of biologically active protein from these structures is usually low [23]. HGCSF can be secreted into the periplasm of E. coli [24,25], low yields are usually obtained using this method. Maltose-binding protein (MBP), and stress-responsive proteins such as peptidylprolyl 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]
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