Efficient expression and purification of human interferon alpha2b in the methylotrophic yeast, Pichia pastoris.
The human interferon alpha2b (hIFN-alpha2b) is the most widely used member of IFNalpha 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-alpha2b has been cloned into the secreting expression organism Pichia pastoris, and the high level expression of hIFN-alpha2b has been achieved. SDS-PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hIFN-alpha2b, 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 1l of the supernatant and its identity to hIFN-alpha2b was confirmed by NH(2)-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-alpha2b for both research and industrial purpose.
Shi
Linmei
L
School of Environmental Engineering, Lishui Vocational & Technical College, Lishui, Zhejiang, PR China. linmeishi2000@126.com <linmeishi2000@126.com>
Wang
Dongming
D
Chan
Weiguang
W
Cheng
Luzi
L
D000595:Amino Acid Sequence; D001681:Biological Assay; D004686:Encephalomyocarditis virus / Q000187:drug effects; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D013058:Mass Spectrometry; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins