Ni nta

High Five cells were grown to 5⋅10⁶ cells/ml and 5 ml were mixed with 5 ml of fresh medium supplemented with penicillin and streptomycin and infected by addition of 200 μl of P2 virus. Cells were harvested after 40 h, pelleted. and lysed by sonication in lysis buffer (50 mM HEPES pH 7.4, 200 mM NaCl, 0.1% Triton X-100, 0.2 mM TCEP). Lysate was cleared by centrifugation (13200 rpm at 30 min, 4°C). 25 μl Ni-NTA (Sigma-Aldrich) or 15 μl Strep-Macro-Prep (IBA AG) were added and sample was incubated on a shaking platform at 4°C for 30 min. After two washes with lysis buffer, the resin was resuspended in SDS-PAGE sample buffer and analyzed by SDS-PAGE.

The cDNA of human Taspase1 was cloned into the pET22b expression plasmid and recombinantly expressed in E. coli strain BL21* (Invitrogen, Darmstadt, Germany). An appropriate MLL substrate was constructed by cloning a cDNA fragment of MLL comprising amino acids 2614–2773 into the pGEX-5T expression vector. The resulting GST–MLL fusion protein (p50) exhibits the CS1 (QVD·GADD) and CS2 (QLD·GVDD) sites for hydrolysis by Taspase1. Expression of the C-terminal His-tagged Taspase1 protein and the N-terminal His-tagged GST–MLL fusion protein (p50) was induced by adding 1 mM IPTG for 4 h, followed by Ni-NTA affinity-purification following the instructions of the manufacturer (Sigma, Munich, Germany). For in vitro cleavage assays, equal amounts of purified Taspase1 and GST–MLL substrate protein (~ 0.5 μg protein) were co-incubated in incubation buffer containing 40 mM Hepes/KOH pH 7.9, 10 mM KCl and 5 mM MgCl₂ for 30 min at 37 °C. Enzyme, substrate and hydrolyzed products were separated on a 12% SDS-PAGE and Coommassie blue-stained for visual inspection.