Renaturation of recombinant human granulocyte colony-stimulating factor produced from Escherichia coli using size exclusion chromatography

Refolding with simultaneously partial purification of recombinant human granulocyte colony-stimulating factor (rhG-CSF) expressed in Escherichia coli (E. coli) by size exclusion chromatography (SEC) is presented in this work. The solution containing the denatured and reduced rhG-CSF in 8.0 mol(.) L-1 urea extracted from the inclusion body was directly injected into a Superdex 75 column and the refolded rhG-CSF was obtained after elution from the column. Several factors, including the concentration of urea in the mobile phase, pH, flow rate, concentration of glutathione, and ratio of GSH to GSSG, concentration of glycerol, sample loading volume, effecting the aim protein refolding were investigated in details. With the selected optimal conditions, the denatured and reduced rhG-CSF was successfully refolded by SEC, and was partially purified during the chromatographic process. When 200 mu L of denatured rhG-CSF at a concentration of 2.3 mg(.) mL(-1) was loaded on the SEC column, rhG-CSF with specific activity of 1.2 x 10(8) IU (.) mg(-1), purity of 83%, and mass recovery of 30% was obtained.