Optimization of Buffer Additives for Efficient Recovery of hGM-CSF from Inclusion Bodies Using Response Surface Methodology

Abstract

Overexpression of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) by Escherichia coli leads to formation of insoluble and inactive proteins, inclusion bodies. The aim of this study was to improve recovery of biologically active hGM-CSF from inclusion bodies. The effect of types, concentrations and pHs of denaturing agents and addition of reducing agents on the yield of inclusion bodies solubilization was evaluated. Next, various conditions were evaluated for refolding hGM-CSF using a two-step design of experiment (DOE) including primary screening by factorial design, and then optimization by response surface design. It was found that hGM-CSF inclusion bodies can be efficiently solubilized with 4 M urea and 4 mM β-mercaptoethanol, pH = 9. A response surface quadratic model was employed to predict the optimum refolding conditions and the accuracy of this model was confirmed by high value of R(2) (0.99) and F-value of 0.64. DOE results revealed that sorbitol (0.235 M), imidazole (97 mM), and SDS (0.09) would be the optimum buffer additives for refolding of hGM-CSF. Following refolding studies, the obtained protein was subjected to circular dichroism which confirmed correct secondary structure of the refolded hGM-CSF. The refolded hGM-CSF exhibited reasonable biological activity compared with standard protein. The approach developed in this work can be important to improve the refolding of other proteins with similar structural features.