Recombinant Expression and Purification of CjRecR

E. coli BL21 (DE3) cells containing the cjRecR-expression plasmid were grown in an LB medium at 37 °C. When the optical density of the culture at 600 nm reached 0.6, the culture was supplemented with isopropyl β-D-1-thiogalactopyranoside (1 mM) to induce cjRecR overexpression. The cells were further cultured at 37 °C for 3 h. The resultant cells were harvested using centrifugation and lysed using sonication in 50 mM Tris, pH 8.0, 200 mM NaCl, and 5 mM β-mercaptoethanol (βME).
The cjRecR protein was first purified using immobilized metal affinity chromatography. The cell lysate containing the hexahistidine-tagged cjRecR protein was incubated with Ni-NTA resin, and the resin was harvested using an Econo-Column (Bio-Rad, Hercules, CA, USA). The cjRecR protein was eluted from the resin using a solution containing 50–500 mM imidazole, 50 mM Tris, pH 8.0, 200 mM NaCl, and 5 mM βME. The eluted cjRecR protein was dialyzed against 20 mM Tris pH, 8.0, and 5 mM βME and then treated with thrombin to remove the hexahistidine tag. Subsequently, the untagged cjRecR protein was purified via anion exchange chromatography using a Mono Q 10/100 column (GE Healthcare, Chicago, IL, USA) with a NaCl gradient (0–500 mM) in 20 mM Tris, pH 8.0, and 5 mM βME.
cjRecO protein was produced in E. coli cells and purified by affinity and ion exchange chromatography as described previously [6 (link)].