Human GCSF was initially purified from a tumor cell line that continuously secreted the protein. When expressed in the methylotrophic yeast Pichia pastoris, hGCSF is secreted in a soluble form; however, the secreted protein is highly aggregated and must be solubilized using high concentrations of denaturants such as guanidine hydrochloride or urea. Consequently, purification of the biologically active form of hGSCF from yeast requires the removal of these denaturants and refolding of the protein. Escherichia coli also produces aggregated hGCSF in inclusion bodies ; however, the overall yield of biologically active protein from these structures is usually low. Alternatively, hGCSF can be secreted into the periplasm of E. coli, although low yields are also usually obtained using this method. Maltose-binding protein, 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. In this study, several new methods of overexpressing soluble hGCSF in the cytoplasm of E. coli were investigated, enabling efficient production of biologically active protein. The following seven N-terminal fusion tags were used: hexahistidine, thioredoxin, glutathione S-transferase, MBP, Nutilization substance protein A, protein disulfide bond isomerase, and the domain of PDI. The MBP, NusA, PDI, and PDIb tags increased the solubility of hGCSF markedly at 30uC. SB431542 Lowering the expression temperature to 18uC also increased the solubility of Trx- and GST-tagged hGCSF, whereas His6-hGCSF was insoluble at both temperatures. The expression level and the solubility of the tag-fused hGCSFs were also tested in the E. coli Origami 2 strain that have mutations in both the thioredoxin reductase and glutathione reductase genes, which may assist the disulfide bond formation in the cytoplasm of E. coli. Simple methods of purifying hGCSF from the PDIba or MBP tagged proteins were developed using conventional chromatographic techniques. In total, 11.3 mg of biologically active hGCSF was obtained from 500 mL of culture. Silver staining indicated that the extracted hGCSF was highly pure and the endotoxin level was very low. The activity of the purified protein was measured using a bioassay with mouse MNFS-60 myelogenous leukemia cells. Many human proteins expressed in prokaryotes such as E. coli are prone to accumulation in IBs. Consequently, time-consuming solubilization and refolding are necessary to generate the purified proteins; processes that are also hampered by low yields, poor reproducibility, and the generation of proteins with low biological activity. When expressed in E. coli, hGCSF is also insoluble, and so to address this problem, this study examined the effect of seven different fusion tags that function as chaperones, as well as the effect of a low expression temperature, on the solubility of hGCSF. The MBP, PDI, PDIb and NusA tags solubilized greater than 70% of the hGCSF fusion protein at 30uC, whereas the solubilities of the Trx.