Pgex 4t

Recombinant proteins tagged with glutathione-S-transferase (GST) were constructed by recombining the insertion sequences of pBluescript into pGEX-4T (GE Healthcare Life Sciences, Pittsburgh, PA) vector plasmids. The pBluescript plasmids were digested using a combination of EcoRI and XhoI or SmaI and XhoI. The inserted DNA fragments were isolated using GeneElute™ Minus EtBr SPIN COLUMNS (Sigma-Aldrich, St. Louis, MO). The insertion sequences were ligated in frame to SmaI- and XhoI-digested pGEX-4T-1 or EcoRI- and XhoI-digested pGEX-4T-3 using Ligation Convenience Kits (Nippon Gene, Toyama, Japan). The ligation mixtures were used to transform ECOS™ competent E. coli JM-109 (Nippon Gene) and appropriate recombinants were confirmed by DNA sequencing. The constructed pGEX recombinants with the correct insertion in the right orientation were then used to transform competent E. coli BL21-RIL-codon-plus (Stratagene). The expression of the GST-fusion proteins was induced by treating the transformed E. coli with 0.1 mM isopropyl-β-D-thiogalactoside for 3 h. The GST recombinant proteins were purified by glutathione-Sepharose column chromatography according to the manufacturer’s instructions (GE Healthcare Life Sciences) and dialyzed against 1 mM Tris–HCl (pH 7.5) and 1 mM EDTA.