Hitrap talon crude 5 ml

Mouse TREX2 gene (Gene ID in NCBI: 24102) was cloned into a pET28a vector and expressed in E. coli BL21-CodonPlus (DE3)-Rosetta strain. Escherichia coli cells were cultured in Luria Broth (LB) medium at 37°C supplemented with 50 mg/ml kanamycin and 35 mg/ml chloramphenicol to an OD₆₀₀ of 0.4–0.6 and then induced with 1 mM isopropyl β-d-1-thiogalactopyranoside at 18°C for 18 h. The cells were harvested through centrifugation and further lysed through sonication in 50 mM Tris–HCl, 300 mM NaCl, pH 8.0. The lysate was clarified through centrifugation at 13 000 rpm at 4°C for 20 min. The supernatant was loaded into an affinity column (HiTrap TALON crude 5 ml, GE Healthcare) and purified by standard protocol. mTREX2 was further purified by an ion-exchange column (HiTrap Q 5 ml, GE Healthcare) and a size-exclusion column (HiLoad 16/60 Superdex 75 prep grade, GE Healthcare). Purified mTREX2 was concentrated to 5–8 mg/ml in 50 mM Tris–HCl, 400 mM NaCl, pH 7.0, and stored at −20°C until use. Active site mutated mTREX2 (H188A) was purified by the same procedure.

The PUFA substrates LA, AA and EPA were purchased from TCI chemicals (Tokyo, Japan). DHA was obtained from Thraustochytrid microalgae (Schizochytrium sp. SH103)^{14 (link)}. The HFA standards 17S-HDHA and resolvin D1-5 were obtained from Cayman Chemical (Ann Arbor, MI, USA). The pfu DNA polymerase premix was purchased from Bioneer Inc. (Daejeon, Korea). Restriction enzymes were supplied by New England Biolabs (Beverly, MA, USA). The epoxide hydrolase (EH) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The E. coli DH5α strain used for gene cloning was purchased from Real Biotech Corporation (Banqiao, Taiwan). The plasmid pET-28a and E. coli BL21 (DE3) were supplied by Novagen (Madison, WI, USA). HiTrap Talon crude (5 mL) and Superdex 200 pg (16/600) were purchased from GE Healthcare (Madison, WI, USA). The SUPELCOSIL LC-DIOL column (25 cm × 3 mm, 5 μm) was obtained from Sigma-Aldrich (St. Louis, MO, USA). The HECTOR-M C18 column (25 cm × 4.6 mm, 5 μm) was purchased from RS Tech (Cheongju, Korea). The CHIRALPAK IB column (25 cm × 4.6 mm, 5 μm) and CHIRALCEL OD-H column (25 cm × 4.6 mm, 5 μm) were obtained from Daicel (Tokyo, Japan). Diaion HP20 resin was obtained from Mitsubishi Chemical (Tokyo, Japan). All solvents for high-performance liquid chromatography (HPLC) analysis were from DaeJung Chemical (Siheung, Korea). All reagents used in this study were extra pure grade.

The wild-type and N-terminal truncated mouse APE1 (mAPE1) genes were cloned into a pET28a vector, respectively, and expressed in E. coli BL21-CodonPlus(DE3)-RIPL strain. E. coli cells were cultured in Luria Broth (LB) medium at 37 °C supplemented with 50 mg/ml kanamycin, 35 mg/ml chloramphenicol and 25 mg/ml streptomycin to an OD₆₀₀ of 0.4–0.6 and then induced by 1 mM isopropyl β-d-1-thiogalactopyranoside at 18 °C for 18 h. The cells were collected through centrifugation at 6,721 g for 30 min at 4 °C and further lysed through sonication in 50 mM Tris-HCl pH 8.0, 300 mM NaCl. The cell debris was clarified through centrifugation at 20,216 g at 4 °C for 30 min and the supernatant was loaded into an affinity column (HiTrap TALON crude 5 ml, GE Healthcare) and purified by standard protocol. Target proteins were further purified by an ion-exchange column (HiTrap™ SP HP 5 ml, GE Healthcare) and a size-exclusion column (HiLoad 16/60 Superdex 75 prep grade, GE Healthcare). Purified wild type or truncated mAPE1 was concentrated to at least 10 mg/mL in 50 mM Tris-HCl pH 7.0, 300 mM NaCl, and stored at −20 °C until use. Active site or R176A/M269A mutants were made by QuikChange lightning site-directed mutagenesis kit (Stratagene) and purified by the same procedure.