For in vitro cleavage assays, wild type and mutant Dronc coding sequences were cloned into pET-28a plasmid to yield 6xHis fusion proteins. Generated plasmids were transformed to BL21(DE3)pLysS competent cells (Promega L1191). 50 ul of bacterial culture was grown at 37°C. Plasmid expression was induced by 0.2 mM IPTG for 3 h at 30°C as described [88 (link)]. Bacterial pellets were lysed with 4 ml of CellLytic B Cell Lysis Reagent (Sigma-Aldrich B7435) after adding 0.2 mg/ml Lysozyme, 50 units/ml Benzonase and 1X protease inhibitor (Roche).
DriceC211A-pET23b plasmid was a kind gift from Dr. Guy Salvesen [53 (link)]. DriceC211A coding sequence was cloned into PT7CFE1-Nmyc plasmid (Thermo Scientific 88863). Myc-DriceC211A protein was generated by using TNT Rabbit Reticulocyte Lysate System (Promega L4610). 4 ul of Myc-DriceC211A protein was incubated with 100 ug of wild-type and mutant 6xHis-Dronc protein in caspase assay buffer (100 mM Hepes pH 7.5, 0.1% CHAPs, 10% sucrose, 10 mM DTT, 50 mM Nacl, 0.5 mM EDTA, protease inhibitor). The reaction was incubated at 30°C for 3 hours [54 (link)] and analyzed by western blotting. Anti-Myc antibody (Santa Cruz SC40) was used at 1:200 concentration.
DriceC211A-pET23b plasmid was a kind gift from Dr. Guy Salvesen [53 (link)]. DriceC211A coding sequence was cloned into PT7CFE1-Nmyc plasmid (Thermo Scientific 88863). Myc-DriceC211A protein was generated by using TNT Rabbit Reticulocyte Lysate System (Promega L4610). 4 ul of Myc-DriceC211A protein was incubated with 100 ug of wild-type and mutant 6xHis-Dronc protein in caspase assay buffer (100 mM Hepes pH 7.5, 0.1% CHAPs, 10% sucrose, 10 mM DTT, 50 mM Nacl, 0.5 mM EDTA, protease inhibitor). The reaction was incubated at 30°C for 3 hours [54 (link)] and analyzed by western blotting. Anti-Myc antibody (Santa Cruz SC40) was used at 1:200 concentration.