Purification of KIF1C Motor Domain

KIF1C motor domain (residues 1–349) was fused to murine KIF5C residues 329–334 followed by 6×His tag (Nitta et al., 2004 (link)) and purified based on Romberg et al. (1998) (link). After expression in Rosetta 2 cells using 0.5 mM isopropyl B-p-thiogalactopyranside for 16 hr at 16°C, cells were suspended in Buffer B (50 mM NaPO₄, pH 7.4, 15 mM imidazole, 250 mM NaCl, 1 mM MgCl₂, 25 µM ATP, protease inhibitors) and disrupted by Emulsiflex C-5 (Avestin, Ottawa, ON). Clarified lysate was incubated with Ni-NTA (Qiagen, Santa Clarita, CA) for 1.5 hr at 4°C and after washing with Buffer B was eluted with Buffer B + 200 mM imidazole. The eluate was diluted fivefold with Buffer C (30 mM Hepes, pH 7.4, 1 mM MgCl₂, 1 mM EGTA, 25 µM ATP) and applied to HiTrap Q FF (GE Healthcare), washed with Buffer C + 100 mM NaCl, before being eluted by gradient to 500 mM NaCl in Buffer C. Binding of the purified components was assayed in the same manner as in vitro translation-synthesized constructs.