Purification of Mutant Human Arrestin

Wild-type human βarr1 was cloned into the pTrcHisB vector with a TEV protease-cleavable N-terminal His₆ and Flag tag. Two mutations were introduced by site-directed mutagenesis: L68C, a finger-loop mutation commonly used in the functional labelling of arrestins^{31 (link)}, and R169E, which disrupts the polar core and predisposes arrestin to activation^{55 (link)}. Arrestin was expressed in BL21 cells. Cells were grown in LB medium supplemented with 100 µg/ml ampicillin at 25 °C. Expression was induced with 30 µM IPTG at a cell optical density at 600 nm (OD₆₀₀) of 0.5. The temperature was lowered to 15 °C and the cells allowed to grow for an additional 20 h. Cells were collected and flash-frozen in liquid nitrogen and stored at –80 °C. Arrestin was purified sequentially by Ni²⁺-affinity chromatography, TEV protease-cleavage of its N-terminal affinity tags, and heparin chromatography, eluting off the heparin column using 1 M NaCl. Purified arrestin was further polished on a Superdex 200 prep grade column (GE Healthcare) equilibrated in 20 mM Tris-Cl⁻ pH 8.0, 0.1 M NaCl, 10% (v/v) glycerol, 0.5 mM DTT. Peak fractions were pooled and concentrated to 20 mg/ml and flash-frozen as aliquots in liquid nitrogen and stored at –80 °C.