Talon affinity chromatography resin

Purification of Flag-DDX3X from HEK293T cells was performed as described previously (Cruciat et al., 2013 (link)). Importantly, during the last washes before elution, the beads were washed with a high-salt buffer (150 mM NaCl) to prevent co-purification of endogenous CK1 protein. Oligomerization control experiments with DDX3X^168-582 revealed that only very low levels of endogenous DDX3X were co-purified by oligomerizing with Flag-DDX3X. For bacterial expression, the pET28a expression vectors were transformed into Rosetta2 cells (DDX3X variants, Novagen) or Lemo21(DE3) (CK1εΔC, NEB). Gene expression was induced by the addition of 0.5 mM IPTG at OD₆₀₀=0.5 and incubation continued for 16 h at 20°C. Cells were lysed by sonication in a lysis buffer consisting of 50 mM Tris-HCl, 200 mM NaCl, 5 mM imidazole, 1 mM TECEP, pH 8.0. The cell lysate was cleared by high-speed centrifugation and subjected to TALON affinity chromatography resin (Clontech) according to the manufacturer's instructions. Proteins for ELISA and FTS were dialyzed in 50 mM HEPES, pH 8 containing protease inhibitor (25 µg/ml aprotinin).

The protein library was prepared in a PUREfrex 2.0 (GeneFrontier Corporation) cell-free protein expression system. The reaction was prepared according to the manufacturer’s recommendations, supplemented with 0.05% Triton X-100 (v/v), and initiated by addition of 3 µg library mRNA. Protein expression was conducted for 4 h at 30°C. The reaction was diluted 10 times with guanidine denaturation buffer (6 M guanidine hydrochloride, 100 mM sodium phosphate, 500 mM NaCl, 0.05% Triton X-100, pH 8) and incubated with 4 µl TALON affinity chromatography resin (Clontech) for 12 h at 25°C. The resin was washed twice with urea denaturation buffer (8 M urea, 100 mM sodium phosphate, 500 mM NaCl, 0.05% Triton X-100, pH 8) and twice with distilled water supplemented with 0.05% Triton X-100. The library was eluted by boiling the affinity matrix in 50 µl of 2% (w/v) aqueous SDS. Eluted fractions were purified from SDS by addition of 5× volumes of ice-cold acetone. The precipitates were centrifuged, washed with 100% acetone and air-dried.