Constructing GST Fusion Protein Plasmids

Expression plasmids of GST fusion proteins were constructed by recombining the cDNA sequences into pGEX-4T vectors (GE Healthcare Life Sciences, Pittsburgh, PA) as previously described [16 , 17 (link), 23 (link)-26 (link), 28 (link)-32 (link), 34 , 39 (link), 42 , 43 (link)]. The pBluescript plasmids containing cDNA inserts were digested with EcoRI and XhoI and separated via agarose gel electrophoresis. The inserted cDNA fragments were isolated using GenElute Minus EtBr Spin Columns (Sigma-Aldrich, St. Louis, MO). Using Ligation Convenience Kits (Nippon Gene, Toyama, Japan), the inserts were properly ligated in frame to EcoRI- and XhoI-digested pGEX-4T-1 or pGEX-4T-3 linearized vectors, which express the recombinant GST-tagged proteins. The ligation mixtures were used to transform ECOS competent E. coli BL-21 cells (Nippon Gene), and appropriate recombinants were confirmed by DNA sequence analysis as well as protein expression. To confirm successful recombination, expression of GST fusion proteins was induced by treating the transformed bacterial clones with 0.1 mM IPTG for 2.5 h, and expressed proteins were electrophoresed through 11% sodium dodecyl sulfate (SDS)-polyacrylamide gels.