Fusion expression and bioactivity comparison of porcine β-defensin-2 and porcine interferon-gamma in Pichia pastoris

Abstract:

In order to study PBD-2 and PoIFNγ, the chimeric gene PBD-2-PoIFNγ was synthesized by overlap extension PCR, and amplified PoIFNγ on the basis of this sequence, then cloned into yeast expression vector pPICZaA separately to get the recombinant plasmid pPICZaA-PBD-2-PoINFγ and pPICZaA-PoINFγ. The recombinant plasmid was digested by Sac I and introduced into Pichia pastoris X33 cells by electroporation. Positive clones were screened and cultivated in BMMY medium containing 0.5% methanol for 72 h. SDS-PAGE and Western blotting analysis showed that the screened recombinant could secrete PBD-2-PoINFγ and PoINFγ separately. The activity of fusion protein was not detected by cytopathic effect inhibition assay and agar diffusion assay, but detected obvious antiviral activity of PoINFγ. The helix and random coil contents was showed vary greatly between PoIFNγ and PBD-2-PoINFγ by circular dichroism analysis. It was speculated that the fusion protein was not correctly folded and may affect the activity of PBD-2-PoINFγ.