Expression and purification of porcine PID1 gene in Escherichia coli

Authors : Huan WANG, Xiaoling CHEN, Zhiqing HUANG, Bo ZHOU, Gang JIA, Guangmang LIU, Hua ZHAO

Pages : 523-527

Doi:10.3906/biy-1403-38

View : 8 | Download : 3

Publication Date : 2014-12-01

Article Type : Research Paper

Abstract :In this study, in order to scale up the production of recombinant porcine phosphotyrosine interaction domain containing 1 insert ignore into journalissuearticles values(pPID1);, a pET-28a insert ignore into journalissuearticles values(+);-pPID1 expression plasmid was constructed and transformed into Escherichia coli Rosetta insert ignore into journalissuearticles values(DE3);. The recombinant pPID1 was then purified and identified by western blotting, and was also analyzed in vitro for its function. The recombinant protein was tagged with only a His6 tag at its C-terminus, which could be conveniently purified by affinity column. The protein could be induced for efficient expression with 0.75 mM IPTG for 8 h at 30 °C, yielding approximately 3 mg/L. In vitro biological activity assay demonstrated that the refolded purified recombinant pPID1 increased 3T3-L1 preadipocyte proliferation. This study provides a reliable technique for the recombinant expression and purification of pPID1 proteins.
Keywords : Porcine PID1, Escherichia coli, expression and purification, identification, 3T3 L1 preadipocytes