Recombinant Expression of RNF219-C Fragment

An MBP-tagged C-terminal fragment of RNF219 (residues 434-726) was produced in Spodoptera frugiperda Sf21 insect cells using the MultiBac baculovirus expression system as previously described^{25 (link)}. In brief, the Sf21cells were grown to a density of 2 × 10⁶ cells/ml at 27 °C in Sf900II medium (Thermo Fisher Scientific), infected with the V1 RNF219-C stock of baculovirus, and harvested 48 h after they stopped dividing. Cells were resuspended in lysis buffer (50 mM HEPES, 500 mM NaCl, pH 7.5) and lysed using a Branson Ultrasonics Sonifier SFX550. The lysate was cleared by centrifugation at 40,000 g for 1 h at 4 °C and filtered through 0.45 µm syringe-driven filters (Millipore). The cleared and filtered lysate was diluted to 250 mM NaCl before it was loaded onto a 5 ml MBPTrap column (Cytiva). The bound protein was eluted in one step with binding buffer (50 mM HEPES, 200 mM NaCl, pH 7.5) supplemented with 30 mM maltose.