Efficient expression and purification of human interferon alpha2b in the methylotrophic yeast, Pichia pastoris

The human interferon alpha2b (hIFN-α2b) is the most widely used member of IFNα family, and it exerts many biological actions including broad-spectrum antiviral effects, inhibition of tumor cell proliferation and enhancement of immune functions. Herein, the cDNA coding for hIFN-α2b has been cloned into the secreting expression organism Pichia pastoris, and the high level expression of hIFN-α2b has been achieved. SDS–PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hIFN-α2b, a 18.8 kDa protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using Source Q ion exchange and Superdex™ 75 size-exclusion chromatography steps. Finally, 298 mg of the protein was obtained in high purity from 1 l of the supernatant and its identity to hIFN-α2b was confirmed by NH2-terminal amino acid sequence analysis. The bioassay of the recombinant protein gave a specific activity of 1.9 × 109 IU/mg. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional hIFN-α2b for both research and industrial purpose.