Protein expression and purification from insect cells

Bacmid recombination and virus production were performed as described previously [54]. A 500-mL culture (SFM4 medium; Hyclone) of Sf21 cells (0.8 × 10⁶ cells/mL) was infected with RILP::Strep-tag II-encoding virus. Cells were harvested by centrifugation at 800g for 5 min. Pellets were resuspended in lysis buffer C (50 mM HEPES, 250 mM NaCl, 1 mM DTT [pH 8.0]) supplemented with EDTA-free cOmplete Protease Inhibitor Cocktail (Roche), sonicated, and cleared by centrifugation at 34,000g for 45 min. RILP::Strep-tag II was purified by batch affinity chromatography using Strep-Tactin Sepharose. The resin was washed with wash buffer C (25 mM HEPES, 250 mM NaCl, 0.1% [v/v] Tween 20, 1 mM DTT [pH 8.0]), and the protein was eluted on a gravity column with elution buffer E. Fractions containing RILP::Strep-tag II were pooled and dialyzed against storage buffer. Glycerol and DTT were added to final concentrations of 10% and 1 mM, respectively, and aliquots were flash-frozen in liquid nitrogen and stored at −80°C.