Purification of Recombinant Murine Proteins

Human his₆-smt-G0S2 constructs were transformed into Escherichia coli BL21(DE3) CodonPlus® cells (Stratagene, La Jolla, CA). Cultures were grown at 37 °C on selective LB medium containing 40 μg/ml kanamycin to an A₆₀₀ of 0.5. Expression was induced by the addition of 0.5 mm isopropyl-β-d-thiogalactopyranoside at 30 °C. After 15 h of induction, cells were harvested, resuspended in sucrose solution (250 mm sucrose, 1 mm EDTA, 1 mm DTT, 20 μg/ml leupeptin, 2 μg/ml antipain, 1 μg/ml pepstatin, pH 7.0), and disrupted by sonication (SONOPLUS ultrasonic homogenizer HD 2070, Bandelin, Berlin, Germany) on ice. After centrifugation at 15,000 × g for 20 min at 4 °C, the supernatants were collected. Protein concentrations were determined as described below. Expression of the murine ATGL-Strep fusion (Strep-mATGL) and His₆-Smt-mCGI-58 in E. coli is described in Refs. 13 (link) and 42 (link), respectively.