Phenyl superose hr5 5

Xenopus laevis adult females (Centre de Ressources Biologiques Xenopes, CNRS, France) were bred and maintained according to current French guidelines in the conventional IBPS aquatic animal facility, with authorization: Animal Facility Agreement: #A75-05-25. All experiments were subject to ethical review and approved by the French Ministry of Higher Education and Research (reference APAFIS#14127-2018031614373133v2).
All reagents, unless otherwise specified, were from Sigma. Okadaic acid (OA), magnetic GSH-beads and Co-beads were purchased from Enzo Life Sciences, Promega and Clontech Laboratories respectively. Uno Q-25 was purchased from Bio-Rad, Mono Q 4.6/100PE, Phenyl-Superose HR5/5 and Superose 12 HR10/30 were from GE Healthcare.

After 96 h flask cultivation of both strains, the culture supernatants obtained after centrifugation (7,000×g, 10 min, 4°C) were filtered using a membrane filter (ADVANTEC^® C045A090C, 0.45 μm, Toyo Roshi Kaisha, Ltd. Japan). One-ml aliquots of filtrates were applied to a TSK-GEL3000SW_XL column (Tosoh) in 50 mM acetate sodium buffer (pH 5.2) containing 0.3 M NaCl at a flow rate of 0.5 ml/min. The absorbance was measured at 280 nm. Fractions containing enzyme activity were concentrated, desalted using Amicon Ultra-15 10000 MWCO (MILLIPORE), and filtered using a syringe filter (ADVANTEC^® DISMIC^®-25cs, 0.2 μm, Toyo Roshi Kaisha, Ltd.). The filtrate (100 μl) was mixed with 900 μl of 50 mM Na-phosphate buffer (pH 7.1) containing 1.3 M ammonium sulfate. The mixture (1 ml) was applied to a hydrophobic interaction column (Phenyl Superose HR 5/5; GE Healthcare UK Ltd.) with a linear gradient (1.2–0 M) of ammonium sulfate in 50 mM Na-phosphate buffer (pH 7.1) at a flow rate of 0.5 ml/min. Fractions containing enzyme activity were concentrated, desalted, and filtered as described above. Protein concentration was measured with a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA).