Refolding and purification of recombinant human interferon-γ expressed as inclusion bodies in Escherichia coli using size exclusion chromatography

A size exclusion chromatography (SEC) process, in the presence of denaturant in the refolding buffer was developed to refold recombinant human interferon-gamma (rhIFN-gamma) at a high concentration. The rhIFN-gamma was overexpressed in E. coli, resulting in the formation of inactive inclusion bodies (IBs). The IBs were first solubilized in 8 M urea as the denaturant, and then the refolding process performed by decreasing the urea concentration on the SEC column to suppress protein aggregation. The effects of the urea concentration, protein loading mode and column height during the refolding step were investigated. The combination of the buffer-exchange effect of SEC and a moderate urea concentration in the refolding buffer resulted in an efficient route for producing correctly folded rhIFN-gamma, with protein recovery of 67.1% and specific activity up to 1.2 x 10(7) IU/mg.