Expression and bioactivity of OSF-1 in Pichia pastoris.
- Author:
Donghong KANG
;
Yan WANG
;
Hongmei ZHANG
;
Xiaoyu FENG
;
Wei CAO
;
Ping WANG
- Publication Type:Journal Article
- MeSH:
Blotting, Western;
Carrier Proteins;
biosynthesis;
Chromatography, Affinity;
Cytokines;
biosynthesis;
Electrophoresis, Polyacrylamide Gel;
Electroporation;
Pichia;
metabolism;
Polymerase Chain Reaction;
Recombinant Proteins;
biosynthesis
- From:
Chinese Journal of Biotechnology
2014;30(2):274-283
- CountryChina
- Language:Chinese
-
Abstract:
In order to research the biologic activity of osteoblast-stimulating factor 1 (OSF-1), the pPIC9K/osf-1 yeast expression vector was constructed to express and purify OSF-1. Firstly, the osf-1 gene sequence was obtained by artificial synthesis and cloned into Pichia pastoris expression vector pPIC9K to generate pPIC9K/osf-1. The recombinant plasmid was linearized by Sac I and transformed into P. pastoris GS115 by electroporation. Recombinant P. pastoris GS115/ pPIC9K/osf-1 was screened by MD and G418-YPD plates and further identified by PCR. The positive P. pastoris was induced with 1% methanol at 25 degrees C for 96 h. The target protein was analyzed by SDS-PAGE showing a special band about 18 kDa. The target protein was successfully purified from the supernatant of the broth using ion exchange chromatography of SP-Sephadex C-50. The purity of target protein was above 98%. Western blotting appeared a good antigenicity of the purified protein. Bioassay results show that the recombinant protein OSF-1 can promote the differentiation and proliferation of osteoblasts MC3T3-E1. We successfully expressed OSF-1 by recombinant P. pastoris for further development of anti-osteoporosis of research and industrial production of OSF-1.
