Preparation of recombinant elongation factor 1 alpha (eEF1α). N-terminal GST fusion proteins of rat eEF1α were over-expressed as full length or sub-domains I, II or III in BL-21 Rosetta
IGF-1 DES by induction with 1 mM IPTG at 37 °C for 2.5 h (GST-eEF1α domain II and pure GST protein) or 0.2 mM IPTG at 16 °C for 16 h (GST-eEF1α full length, domains I and III). All the pGEX vectors were kindly donated by Gang Liu (Albert Einstein College of Medicine). Recombinant GST protein alone was also purified and used as a control. Cell pellets were re-suspended in pre-cooled resuspension buffer (1× PBS, 5 mM β-mercaptoethanol), disrupted by sonication and the suspension centrifuged at 30,000g at 4 °C for 30 min. The supernatant fluid was filtered (0.2 μm) and incubated with glutathione Sepharose? 4B (GE Healthcare). After washing, bound proteins were eluted with 10 mM reduced glutathione, 50 mM Tris
pH 8.0, 50 mM NaCl, 10 mM β-mercaptoethanol. The purity of eluted protein was examined by SDS–PAGE and aliquots stored with 15% glycerol at ?80 °C.
