Human reticulocyte 15-lipoxygenase-1 (15-hLO-1) and human platelet 12-lipoxygenase (12-hLO) were expressed as N-terminally, His6-tagged proteins and purified to greater than 90% purity, as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.9 (link), 35 (link) Human 5-lipoxygenase (5-hLO) in a pET21 expression vector was received from the Tatulian Lab (University of Central Florida).36 (link) It was transformed into E. coli BL21-DE3 and grown overnight in LB medium (50 mL) with 0.1 mg/mL Ampicillin at 25 °C. Next, the 50 mL growth mixture was separated into 7 × 2 L flasks of media and grown at 30 °C until the OD600 reached 0.6. The cells were then induced with 0.25 mM IPTG, followed by lowering the temperature to 18 °C. The cells were harvested 16 – 20 hours post-induction by pelleting at 5,000 g (10 min) and stored at −80 °C until purification.
The frozen cell pellet was resuspended in buffer A (10 mM β-mercaptoethanol, 0.1 mM EDTA, and 50 mM triethanolamine (pH 7.3)) and placed on ice. Cells were lysed using a Power Laboratory Press and cleared by spinning at 40,000 g (25 min). Proteins in the supernatant were precipitated at 50% saturated ammonium sulfate and spun at 40,000 g (25 min). Proteins in the salt pellet can be stored under liquid nitrogen with negligible loss of 5-hLO activity for over 6 months. The salt pellets were resuspended in buffer A for use in activity assays.
The frozen cell pellet was resuspended in buffer A (10 mM β-mercaptoethanol, 0.1 mM EDTA, and 50 mM triethanolamine (pH 7.3)) and placed on ice. Cells were lysed using a Power Laboratory Press and cleared by spinning at 40,000 g (25 min). Proteins in the supernatant were precipitated at 50% saturated ammonium sulfate and spun at 40,000 g (25 min). Proteins in the salt pellet can be stored under liquid nitrogen with negligible loss of 5-hLO activity for over 6 months. The salt pellets were resuspended in buffer A for use in activity assays.
