TAILIEUCHUNG - Expression and purification of porcine PID1 gene in Escherichia coli
In this study, in order to scale up the production of recombinant porcine phosphotyrosine interaction domain containing 1 (pPID1), a pET-28a (+)-pPID1 expression plasmid was constructed and transformed into Escherichia coli Rosetta (DE3). The recombinant pPID1 was then purified and identified by western blotting, and was also analyzed in vitro for its function. | Turkish Journal of Biology Research Article Turk J Biol (2014) 38: 523-527 © TÜBİTAK doi: Expression and purification of porcine PID1 gene in Escherichia coli Huan WANG, Xiaoling CHEN*, Zhiqing HUANG, Bo ZHOU, Gang JIA, Guangmang LIU, Hua ZHAO Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu, Sichuan, P. R. China Received: Accepted: Published Online: Printed: Abstract: In this study, in order to scale up the production of recombinant porcine phosphotyrosine interaction domain containing 1 (pPID1), a pET-28a (+)-pPID1 expression plasmid was constructed and transformed into Escherichia coli Rosetta (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 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. Key words: Porcine PID1, Escherichia coli, expression and purification, identification, 3T3-L1 preadipocytes 1. Introduction Meat quality in domestic animals is an important economic trait. The quality and the value of the carcass in domestic meat animals are reflected as the ratio between protein and fat content in a carcass. Preadipocytes and adipocytes are important in establishing the overall fatness of a carcass (Hausman et al., 2009). Therefore, it is necessary to identify candidate genes that might contribute to fat .
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