Formation of dimers in refolding of recombinant human interferon-γ by ion-exchange chromatography

Abstract

The refolding process was performed by gradually decreasing the concentration of urea in the buffer after the denatured rhIFN-γ proteins had been bound onto the ion-exchange gel SP Sepharose Fast Flow. Results showed that the denatured rhIFN-γ was refolded efficiently by ion-exchange chromatography with a urea gradient and the purity of the refolded rhIFN-γ was up to 95%. The protein recovery was 54% and specific activity of rhIFN-γ was up to 7.5×105 IU·mg-1. The chromatogram of size exclusion chromatography with Superdex 75 indicated that the refolded rhIFN-γ didn't form any aggregates after renaturation. The conformation of refolded rhIFN-γ was close to the native dimer as shown by fluorescence spectrophotometer characterization.

Keywords

• recombinant human interferon-<italic>γ</italic>
• inclusion body
• renaturation
• ion-exchange chromatography
• fluorescence spectrum analysis