RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination

Nila Roy Choudhury; Gregory Heikel; Maryia Trubitsyna; Peter Kubik; Jakub Stanislaw Nowak; Shaun Webb; Sander Granneman; Christos Spanos; Juri Rappsilber; Alfredo Castello; Gracjan Michlewski

The full-length human TRIM25 and RBD mutant were cloned into pET30a, which was transformed into BL21 gold cells. Bacteria was grown in Superbroth in the presence of kanamycin at 37°C until the OD 600 reached 0.6. The bacteria were induced by 0.5 mM IPTG and incubated for 24 hours at 30°C with agitation. Pellets were lysed with a cell disruptor in lysis buffer (20 mM NaPPi pH 7.4, 500 mM NaCl, 5 mM MgCl2, protease inhibitor, DNase and RNase or benzonase), and lysates were loaded onto an IMAC HiTrap 1-ml FF column in binding buffer (20 mM NaPPi pH 7.4, 500 mM NaCl, 10 mM Imidazole) and eluted with increasing amounts of elution buffer (20 mM NaPPi pH 7.4, 500 mM NaCl, 500 mM Imidazole). Protein fractions were further purified with a gel exclusion column (Superdex 200 30/100 GL or HiLoad Superdex 200 16/60) in gel exclusion buffer (10 mM Tris pH 8.0, 300 mM NaCl, 2 mM DTT). Selected fractions were pooled and concentrated using Vivaspin 5 30000 MWCO (Sartorius Stedim UK Ltd.). His-Lin28s was produced and purified as specified earlier (Nowak et al., 2016) .