tailieunhanh - Heterologous expression and purification of porcine fat mass and obesity-associated gene in Escherichia coli

A porcine fat mass and obesity-associated gene (pFTO) was cloned into the expression vector pET30a(+) and heterologously expressed in Escherichia coli. The effects of isopropyl β-D thiogalactopyranoside (IPTG) concentration and induction time on the expression of recombinant pFTO were assessed. | Turkish Journal of Biology Research Article Turk J Biol (2015) 39: 217-222 © TÜBİTAK doi: Heterologous expression and purification of porcine fat mass and obesity-associated gene in Escherichia coli Bo ZHOU, Xiaoling CHEN*, Zhiqing HUANG, Gang JIA, Guangmang LIU Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu, Sichuan, . China Received: Accepted: Published Online: Printed: Abstract: A porcine fat mass and obesity-associated gene (pFTO) was cloned into the expression vector pET30a(+) and heterologously expressed in Escherichia coli. The effects of isopropyl β-D-thiogalactopyranoside (IPTG) concentration and induction time on the expression of recombinant pFTO were assessed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the molecular mass of the recombinant pFTO protein was about 56 kDa, and the recombinant protein could be induced efficiently in E. coli BL21 by the addition of mM IPTG for 4 h at 30 °C. The recombinant pFTO fusion protein was purified using Ni-IDA affinity chromatography with a yield of about µg/mL protein. The protein was further confirmed by western blot analysis. An in vitro biological activity assay demonstrated that the refolded purified 2 µg/mL recombinant pFTO protein increased 3T3-L1 preadipocyte proliferation. The present work should be useful for the production of sufficiently large amounts of recombinant pFTO protein for further functional analysis. Key words: Porcine FTO, expression, Escherichia coli, purification, identification, 3T3-L1 preadipocytes 1. Introduction Intramuscular fat (IMF), located throughout skeletal muscle (Wood, 1990), is a key factor that affects the flavor, juiciness, and tenderness of meat. The amount of intramuscular fat is expected to be favorably correlated