Atp agarose column

Human recombinant 5-LO was expressed
in Escherichia
coli (BL21, DE3) transformed with the pT3-5-LO plasmid
at 30 °C overnight, as described before.^{26 (link)} Cells were lysed using lysis buffer and homogenized by sonification
(3 × 15 s, Branson Sonifier 250, Branson Ultrasonics Corporation).
5-LO was then purified from 40,000g supernatant (20
min, 4 °C) using an ATP agarose column (Sigma-Aldrich), diluted
with PBS (Dulbecco’s formula, pH 7.4) buffer containing 1 mM
EDTA, and immediately used for 5-LO activity assays.

Human recombinant 5-LOX was expressed in E. coli BL21 (DE3) transformed with pT3-5LO plasmid, and purification of 5-LOX was performed as described before [37 (link)]. Briefly, E. coli were lysed in 50 mM triethanolamine/HCl pH 8.0 plus EDTA (5 mM), soybean trypsin inhibitor (60 µg/mL), phenylmethanesulphonyl fluoride (1 mM), dithiothreitol (1 mM), and lysozyme (1 mg/mL) and then sonicated (3 × 15 s). The homogenate was then centrifuged at 40,000× g for 20 min at 4 °C. 5-LOX in the supernatant was partially purified by affinity chromatography on an ATP-agarose column (Sigma Aldrich, Munich, Germany). Semi-purified 5-LOX was diluted in PBS containing EDTA (1 mM) and immediately used for activity assays.
5-LOX (0.5 µg/mL) was pre-incubated with the test items for 15 min at 4 °C. 5-LOX product formation was initiated by addition of 2 mM CaCl₂ plus 20 µM arachidonic acid. After 10 min at 37 °C, the reaction was terminated by adding 1 mL ice-cold methanol. Formed 5-LOX metabolites (all-trans isomers of LTB₄ and 5-hydro(peroxy)eicosatetraenoic acid (H(P)ETE)) were analyzed by RP-HPLC as described [38 (link)].

Human 5LO was expressed in E. coli BL21-(DE3) (NEB) transformed with pT3-5LO [25 (link)] and purified with ATP Agarose column (Sigma-Aldrich Co.) followed by gel filtration [24 (link)]. As an outline, the post induction cells were resuspended in lysis buffer (100 mM Tris-HCl pH 7.5, 100 mM NaCl, 2 mM EDTA, 1mM FeSO₄, 2 mM TCEP) with protease cocktail inhibitor and lysozyme 0.5mg/ml) followed by sonication (5 x 15s). After clarifying the lysed cells, the supernatant was subjected to ammonium sulfate precipitation and the precipitate (30–60% saturation) was resuspended in lysis buffer. The sample was applied to an ATP Agarose column and the recombinant 5LO was eluted using 20mM ATP in lysis buffer with 10 μM FeSO₄ and 20 μg/ml catalase [26 (link)]. The buffer was changed to 20 mM Tris pH 7.5, 100 mM NaCl, 2 mM EDTA, 1mM FeSO₄, 2 mM TCEP, 20 μg/ml catalase. The concentration of 5LO in elutaes was determined by Bradford assay [27 (link)]. Also MSP1E3D1 (with a histidine tag as well as a TEV protease cleavage site, from Addgene, MA, USA) [21 (link)] was expressed in E. coli (BL21-DE3). The concentration of purified MSP1E3D1 was determined by the absorbance at 280 nm (ε = 29910cm^-1M^-1).