Secretory Expression of recombinant human Interferon-alpha2b in methilotropic yeast Pichia pastoris

Abstract

Interferon is a cytokine secreted by eukaryotic cells as a response to virus, bacteria, and different type of macromolecules exposure. This research was purposed to obtain human interferon-alpha2b (hIFN-α2b) protein in methylotropic yeast Pichia pastoris (P. pastoris). The isolated and amplified open reading frame (ORF) was produced as 522 bps that corresponding to its theoretical hifn-α2b ORF size. The ORF was cloned into pPICZαB shuttle vector and transformed into Escherichia coli (E. coli). Nucleotide sequence analysis confirmed that the recombinant plasmid contained correct sequence of hifn-α2b ORF. The recombinant plasmid pPICZαB-hifn-α2b was linearized by using Bstx1 enzyme and transformed into P. pastoris genome. High expression screening of transformants that performed by using 2000 µg/mL zeocin as a marker resulted in thirteen transformants. All transformants produced 24.05 kDa protein bands in extracellular compartment by using 0.5% methanol as inducer. Slot blot analysis confirmed that the band was hIFN-α2b with the highest expression achieved by clone number 12. To conclude, hIFN-α2b protein was successfully obtained as secretory protein with 24.05 kDa in size containing polyhistidine tag and c-myc epitope in its C terminus.