Expi expression medium

His-mATGL and its point mutants were recombinantly expressed in Expi293F™ cells (Thermo Fisher Scientific, Waltham, USA). The cells were cultivated in Expi Expression Medium (Thermo Fisher Scientific, Waltham, USA) at standard conditions (37°C, 95% humidified atmosphere, 7% CO₂). Cell density was determined in a CASY Cell Counter and Analyzer System (OMNI Life Science, Bremen, Germany). Cells in a total culture volume of 10 ml were transfected with 10 μg of plasmid DNA using the ExpiFectamineTM 293 Transfection Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Two days after transfection, cells were harvested by centrifugation at 500 g and 4°C for 5 min. The cell pellet was washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄). Cells were disrupted immediately after harvesting by sonication (Sonopuls GM3100 equipped with a MS-72 tip; Bandelin, Berlin, Germany) in sucrose solution (250 mM sucrose, 1 mM EDTA, 1 mM DTT, 20 μg/ml leupeptin, 2 μg/ml antipain, and 1 μg/ml pepstatin) on ice. The homogenate was centrifuged at 1,000 g and 4°C for 20 min. The postnuclear fraction was collected and protein concentration was determined by the Bradford protein assay (Bio-Rad Laboratories, Hercules, USA) using (BSA) as standard.

For suspension cell culture, EXPI293F cells were maintained in suspension cultures in Expi Expression Medium (Thermo Fisher Scientific) at 37 °C, 8% CO₂ and high humidity on a shake platform set to 125 rpm. As per manufacturer recommendation, cell density was maintained at 0.3–8 × 10⁶ cells/mL in polycarbonate vented Erlenmeyer flasks (Corning) containing a medium volume of 1/4–1/3 of the total volume of the flask. For stable cell lines, medium containing Geneticin or Zeocin (at the appropriate concentration detailed in Section 2.6) was replenished every 3–5 days.