Expression and purification of CueR

For construction of the plasmid pET21a-CueR, the E. coli CueR gene was PCR-amplified from E. coli genomic DNA and ligated into pET21a vector with C-terminal 6×His-tag using Nde I and Xho I restriction sites. Mutations in the plasmid were introduced by oligos following the Quickchange-site-directed mutagenesis protocol (Stratagene).

All constructs were transformed into chemically competent E. coli BL21(DE3) cells. The cells were grown in LB medium with 100 μg/mL ampicillin at 37°C to the OD₆₀₀ value of 0.6, and protein expression was induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 20°C for 16 h. The harvested cells were suspended in lysis buffer containing 50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole, 5% (v/v) glycerol and lysed via sonication. The lysate was then centrifuged at 80,000 g for 1 h. The recombinant CueR protein was purified through a 5-mL HisTrap column (GE Healthcare) and 5-mL HiTrap Heparin column (GE Healthcare) and further loaded onto a gel filtration column, 120-mL HiLoad 16/600 Superdex 200 in a buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM DTT. The final sample was aliquoted, flash-frozen in liquid nitrogen, and stored at −80°C until use.