Cell lysates containing full-length Kap121 were applied to a HisTrap HP column (GE Healthcare) and washed with the His lysis buffer for ~15 column volumes, followed by a secondary wash with lysis buffer supplemented with 500 mM NaCl. Last, His-Kap121 was eluted with 20 mM Hepes (pH 7.5), 100 mM NaCl, 250 mM imidazole, 5% (v/v) glycerol, and 5 mM BME. After TEV protease cleavage overnight at 4°C, affinity-purified Kap121 was loaded onto the HiTrap Q column (GE Healthcare) and purified via a salt gradient from 100 to 600 mM NaCl. Kap121 was finally purified in a storage buffer using the Superdex 200 16/60 gel filtration column, concentrated to 25 to 30 mg/ml, flash-frozen in liquid nitrogen, and stored at −80°C.

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