Ni sepharose column his trap hp

The cDNA encoding human mitochondrial MTHFD2 (isoform 2; NCBI Reference Sequence: XP_006711987.1) was cloned into pET15b vector with a His-tag in N-terminal and transformed into E. coli BL21 (DE3). The transformed cells were grown in LB-medium containing 100 μg/mL ampicillin at 37 °C overnight. The overnight cultures were inoculated as 1/100 into 1 L of LB-medium containing Ampicillin (100 μg/ml). Cultures were grown at 37 °C OD600 of 0.6–0.8, protein expression was induced by addition of 0.2 mmol/L isopropyl β-d-1-thiogalactopyranoside. Cells were grown at 22 °C overnight, then the bacteria were harvested by centrifugation and washed in PBS. Cells were first dissolved in 30 ml binding buffer (20 mm Tris-HCl pH 8.0, 500 mm NaCl, and 30 mm imidazole), PMSF (0.5 mm) was added just before lysis. Then bacteria were lysed through high pressure disruption by a low temperature Ultra-High-Pressure Continuous Flow Cell Disrupter (JN-3000 plus), the cell lysate was centrifuged at 18,000 rpm for 1 h at 4 °C. The supernatant was loaded onto a 1 ml Ni-Sepharose column His Trap-HP (GE Healthcare) by a peristaltic pump and gradient eluted by an ÄKTA FPLC system (GE Healthcare) with elution buffer (20 mm Tris-HCl pH 8.0, 500 mm NaCl, and 500 mm imidazole). Fractions with eluted MTHFD2 were determined by western blot.

DNA fragments of human ALDH1L2 and its K70Q mutant were cloned into pET28a-sumo plasmids, and were expressed in Escherichia coli BL21 (DE3) using IPTG (Sigma-Aldrich, 1 mM) induction method. These bacteria were harvested and lysed. After centrifugation for 17,000 g for 1 h, the supernatants were collected and loaded onto a 1 ml Ni-Sepharose column His Trap-HP (GE HealthCare) by a peristaltic pump and gradient eluted by an ÄKTA FPLC system (GE HealthCare) with elution buffer (20 mM Tris–HCl pH 8.0, 500 mM NaCl, and 500 mM imidazole).