Expression and purification of recombinant histones and refolding of histone octamers

Human H2A and H2B and Xenopus laevis H3 and H4 was purified as described (Luger et al., 1999). Briefly, H2A, H2B, and H3 were expressed in BL21 (DE3) pLysS cells while H4 were expressed in BL21 cells using LB media. The histones were purified from inclusion bodies using a Dounce glass/glass homogenizer. After solubilization of the inclusion bodies, a three-step dialysis against urea dialysis buffer (7 M urea, 100 mM NaCl, 10 mM Tris, pH 8, 1 mM EDTA, and 5 mM β-mercaptoethanol) was performed. The sample was then applied to a HiTrap Q anion exchange column and a HiTrap SP cation exchange column (GE Healthcare). Histones were eluted from the HiTrap SP column using a linear gradient from 100 mM to 1 M NaCl in 7 M urea, 10 mM Tris, pH 8, 1 mM EDTA, and 1 mM DTT. Purified recombinant histones were dialyzed against water containing 5 mM β-mercaptoethanol before lyophilization and storage at −80°C.

To generate histone H3 phosphorylated at threonine 3 by native chemical ligation, histone H3 lacking residues 1–31, containing a threonine-to-cysteine substitution at position 32 and a cysteine-to-alanine substitution at position 110 (H3Δ1–31 MT32C C110A), was expressed and purified as described above. Native chemical ligation reactions with H3Δ1–31 MT32C C110A and the N-terminal H3 peptide ARTPhKQTARKSTGGKAPRKQLATKAARKSAPA containing a C-terminal benzyl thioester (Peptide Protein Research) were performed in 6 M guanidine HCl, 250 mM sodium phosphate buffer, pH 7.2, 150 mM 4-mercaptophenylacetic acid, and 50 mM tris(2-carboxyethyl)phosphine for 72 h at room temperature with constant agitation. Reactions were dialyzed three times against 7 M urea, 100 mM NaCl, 10 mM Tris, pH 8, 1 mM EDTA, and 1 mM DTT. Ligated full-length H3T3Ph histone was separated from unligated truncated histone through cation exchange chromatography on a monoS column (GE Healthcare) and then dialyzed against water containing 5 mM β-mercaptoethanol before lyophilization and storage at −80°C. To generate tailless H3, we used the H3Δ1-31 MT32C C110A histone H3 to generate the octamers.

Histone octamers were obtained as previously described (Luger et al., 1999). Briefly, lyophilized histones were resuspended in unfolding buffer (7 M guanidine HCl, 20 mM Tris, pH 7.5, and 10 mM DTT) and mixed to equimolar ratios. The histone mix was then dialyzed three times against 500 ml of refolding buffer (10 mM Tris, pH 8, 2 M NaCl, 1 mM EDTA, and 5 mM β-mercaptoethanol). The octamers were obtained by running the histone mix on a size-exclusion chromatography column (Superdex 200 increase 10/300, GE Healthcare) preequilibrated with refolding buffer and stored at −80°C.