Histones expression and purification

All the histones were expressed using pET11a plasmid as expression vector for BL21 (DE3) Star E. coli strain. Cultures were grown in 150 ml of LB/ampicillin (100 μg/ml) media at 37°C overnight. Then, 4 l of LB/ampicillin (25 μg/ml) were inoculated (1:100 dilution) and incubated under the same conditions until reaching an OD (at a wavelength of 600 nm) of 0.4. Protein expression was induced with 1 mM IPTG at 37°C for 2–4 h. Cells were ruptured by sonication in ice and benzonase (Merck-Millipore, Madrid, Spain) was added (20 U/ml) to degrade nucleic acids.

The extraction and purification protocol was adapted from (47). Briefly, the cellular pellet was resuspended in 10 ml SA10X buffer, containing sodium acetate 400 mM pH 5.2, EDTA 10 mM pH 8, methionine 100 mM, and 18 g of urea powder, 0.58 mg of NaCl were added, and completed up to a 50 ml final volume containing β-mercaptoethanol 5 mM. After sonication, the filtered supernatant was centrifuged to remove insoluble cellular debris.

Proteins were purified by ion exchange chromatography, using a column tandem Sepharose Q–Sepharose SP (top–bottom) (GE-Healthcare Life Sciences, Barcelona, Spain) pre-equilibrated in buffer SAU200 (sodium acetate 40 mM, pH 5.2, urea 6 M, EDTA 1 mM pH 8, β-mercaptoethanol 5 mM, methionine 10 mM, NaCl 200 mM). After a 2 ml/min injection, SP column was removed and the protein was eluted from Q column using a gradient with elution Buffer (sodium acetate 40 mM, pH 5.2, urea 6 M, EDTA 1 mM pH 8, β-mercaptoethanol 5 mM, methionine 10 mM, NaCl 1 M. Refolding of the protein was accomplished by dialyzing the protein-containing fractions with deionized water, followed by lyophilization for storage. Purity and homogeneity were checked by SDS-PAGE. Potential DNA contamination was always assessed by the ratio of UV absorption at 260 nm versus absorption at 280 nm. The extinction coefficients respectively at 280 nm used for protein concentration determination are 4050, 6070, 4040 and 5400 M⁻¹ cm⁻¹ for H2A, H2B, H3 (wild-type and variants) and H4, respectively.