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

Abstract

A porcine fat mass and obesity-associated gene (pFTO) was cloned into the expression vector pET30a(+) and heterologouslyexpressed in Escherichia coli. The effects of isopropyl β-D-thiogalactopyranoside (IPTG) concentration and induction time on theexpression of recombinant pFTO were assessed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the molecularmass of the recombinant pFTO protein was about 56 kDa, and the recombinant protein could be induced efciently in E. coli BL21 by theaddition of 0.75 mM IPTG for 4 h at 30 °C. The recombinant pFTO fusion protein was purifed using Ni-IDA afnity chromatographywith a yield of about 1.8 µg/mL protein. The protein was further confrmed by western blot analysis. An in vitro biological activity assaydemonstrated that the refolded purifed 2 µg/mL recombinant pFTO protein increased 3T3-L1 preadipocyte proliferation. The presentwork should be useful for the production of sufciently large amounts of recombinant pFTO protein for further functional analysis.