Isopropyl β d 1 thiogalactopyranoside iptg

Recombinant E. coli Rosetta cells containing the OsRIP1-HIS6 constructs in the pET21a vector were grown overnight in 5 mL LB supplemented with 25 µg/mL carbenicillin and 25 µg/mL chloramphenicol at 37 °C on a rotary shaker at 185 rpm. The next morning, the E. coli pre-cultures were used to inoculate cultures of 300 mL LB supplemented with 25 µg/mL carbenicillin and 25 µg/mL chloramphenicol and these cultures were grown at 37 °C on a rotary shaker (185 rpm). When an OD600nm of 0.6–0.8 was reached, recombinant protein expression was induced with 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (MP Biomedicals, Valiant, Yantai, Shandong, China). After induction, cultures were grown for 72 h at 14 °C on a rotary shaker at 185 rpm. Subsequently cells were harvested by centrifugation at 8000 rpm for 20 min and the cell pellets were pooled and kept overnight at −20 °C. The next day the cell pellet was thawed and resuspended in phosphate buffer (PB; 0.02 M NaH₂PO₄∙2H₂O, 0.23 M Na₂HPO₄) at pH 8 containing 1 mg/mL lysozyme to facilitate cell lysis. The solution was sonicated and afterwards centrifuged at 4 °C for 45 min to remove cell debris and the insoluble protein fraction. The soluble fraction was used for recombinant OsRIP1 protein purification.