Inducible and constitutive expression of fip-fve from Flammulina velutipes in Pichia pastoris GS115

Abstract:

We transformed the fip-fve gene into Pichia pastoris GS115 for inducible and constitutive expression to obtain feasible bioactvie recombinant Fip-fve. The fip-fve gene was cloned from Flammulina velutipes fruting body by PCR and ligated to pPIC9 to construct inducible expression vector pPIC9-FIP-fve, and promotor pgap was used to replace the paox1 to construct constitutive expression vector pPIC9-PGAP-FIP-fve. These two vectors were used to transform P. pastoris by PEG method. The fip-fve was expressed after histamine-absence screening and yeast colony PCR. The inducible expression level reached 158.2 mg/L at the fourth day and the constitutive expression level was 46.3 mg/L and 29.5 mg/L using glucose and glycerol, respectively. The SDS-PAGE and Western blot both proved the correctness of rFip-fve, and the hemagglutination test indicats the rFip-fve's bioactivity.

Key words:Flammulina velutipes, immunomodulation, Pichia pastoris, constitutive expression