Purification of p31comet:C-Mad2:Cdc20 Complex

A ternary complex composed of p31^comet :C-Mad2:Cdc20 was purified by Ni-NTA column chromatography starting with a co-lysate of C-Mad2:MBP-Cdc20 co-expressing Hi-5 insect cells 28 (link) with p31^comet expressing BL21 Star (DE3) cells, followed by TEV cleavage and gel filtration (Superdex 200 10/300 column). Purified TRIP13^Q253E was incubated with equimolar amounts of the p31^comet :C-Mad2:Cdc20 complex (accounting for hexamerization of TRIP13) in an assembly reaction buffer (50 mM HEPES pH 8.0, 10 mM MgCl₂ and 5 mM ATPγS) for 45 min at 23 °C. The 50 μL assembly reaction was injected into a Superdex 200 5/150 GL column fitted to an ÄKTAmicro (GE Healthcare) with a running buffer containing 20 mM HEPES pH 8.0, 300 mM NaCl, 10 mM MgCl₂, 0.5 mM TCEP and 0.3 mM ATPγS. The complex eluted in a peak fraction at a concentration of 0.3 mg/mL and was used to prepare cryo-EM grids.