The human interferon alpha2b (hIFN-alpha 2b) is the most widely used member of IFN alpha 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-alpha 2b has been cloned into the secreting expression organism Pichia pastoris, and the high level expression of hIFN-alpha 2b has been achieved. SDS-PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hIFN-alpha 2b, 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(TM) 75 size-exclusion chromatography steps. Finally, 298 mg of the protein was obtained in high purity from 11 of the supernatant and its identity to hIFN-alpha 2b was confirmed by NH2-terminal amino acid sequence analysis. The bioassay of the recombinant protein gave a specific activity of 1.9 x 10(9) IU/mg. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional hIFN-alpha 2b for both research and industrial purpose. (C) 2007 Elsevier Inc. All rights reserved.