Recombinant Histone Purification Protocol

Histones were expressed and purified as described in Kilic et al. (2015) (link). Briefly, individual wild-type human histones were cloned into pet15b plasmid vectors and expressed in BL21 DE3 plysS cells. Cells were grown in LB media containing 100 μg/mL ampicillin and 35 μg/mL chloramphenicol at 37°C until the OD₆₀₀ reached 0.6. Expression was induced by IPTG addition to a final concentration of 0.5 mM. After 3 h expression, cells were harvested by centrifugation and resuspended in lysis buffer (20 mM Tris pH 7.5, 1 mM EDTA, 200 mM NaCl, 1 mM βMe, Roche protease inhibitor) and frozen. Cells were lysed by freeze-thawing and sonication. Inclusion bodies were harvested by centrifugation. The inclusion body pellet was washed once with 7.5 mL of lysis buffer containing 1% Triton and once without. Inclusion body pellets were resolubilized in resolubilization buffer (6 M GdmCl, 20 mM Tris pH 7.5, 1 mM EDTA, 1 mM βMe) and dialyzed into urea buffer (7 M urea, 10 mM Tris, 1 mM EDTA, 0.1 M NaCl, 5 mM 1 mM βMe, pH 7.5). Histones were purified by cation exchange chromatography using a HiTrap SP HP 5 mL column (GE Healthcare). Fractions were analyzed by SDS-PAGE and pooled, followed by dialysis into water and lyophilization. Final purification was performed by preparative RP-HPLC. Purified histones were lyophilized and stored at −20°C until used for octamer refolding.