Inducible and constitutive expression of fip-fve from Flammulina velutipes in Pichia pastoris GS115.
- Author:
Jingwei LIN
;
Jia JIA
;
Ming ZHONG
;
Lijing CHEN
;
Haoge LI
;
Zhifu GUO
;
Mingfang QI
;
Lixia LIU
;
Tianlai LI
- Publication Type:Journal Article
- MeSH:
Electrophoresis, Polyacrylamide Gel;
Flammulina;
chemistry;
Fungal Proteins;
biosynthesis;
Genetic Vectors;
Pichia;
metabolism;
Polymerase Chain Reaction;
Promoter Regions, Genetic;
Recombinant Proteins;
biosynthesis
- From:
Chinese Journal of Biotechnology
2014;30(3):464-471
- CountryChina
- Language:Chinese
-
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 blotting both proved the correctness of rFip-fve, and the hemagglutination test indicats the rFip-fve's bioactivity.
