On-column refolding of consensus interferon at high concentration with guanidine-hydrochloride and polyethylene glycol gradients.

Dilution refolding of consensus interferon (C-IFN) had a limit on final concentration not exceeding 0.1 mg ml(-1) in order to achieve specific activity of 2.2x10(8) U mg(-1). Addition of polyethylene glycol (PEG) only gave a marginal improvement on the specific activity. Hydrophobic interaction chromatography (HIC) was tried but a simple step-wise elution could not refold the protein. Successful refolding was achieved by gradient elution with the decreasing of guanidine-hydrochloride (guanidine-HCl) concentration. The column was packed with a commercially available HIC medium that was designed for protein separation. Polyethylene glycol was found to possess better effect on the column than in the dilution for promotion of correct refolding, especially in gradient mode. A novel dual-gradient strategy, consisting of decreasing guanidine-HCl concentration and increasing PEG concentration, was developed to enhance the refolding yield. Denatured C-IFN was allowed to adsorb and elute from the HIC column through a gradually changed solution environment. Compared with dilution refolding, the gradient HIC process, in the presence of PEG, gave about 2.6-folds of increase in specific activity, 30% increase in soluble protein recovery. Partial purification was also achieved simultaneously.

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