Thrombin digestion

Histone octamers were purchased from Abcam (ab45275) and purified by gel filtration (GE, S200). The expression plasmids yNap1 and yAsf1 were gifts from Bradley R. Carins (University of Utah) and Jessica K. Tyler (University of Texas) respectively. yNap1 (Nap1) was expressed and purified by Ni-NTA column and ion-exchange chromatography [50 (link)]. yAsf1 (Asf1) was expressed and purified by GST column and ion-exchange chromatography, and the GST tag was removed using thrombin digestion (Sigma) [51 (link)]. All the DNA molecules (211-bp, 433-bp, 614-bp and 836-bp) were prepared by PCR using pBR322 (NEB) as the template. The primer sequences are listed in Table 2.

Recombinant TtSmtB was purified to homogeneity using the procedure already described, consisting of thermo-precipitation of the E. coli BL21-Codon Plus (DE3) RIL/TtSmtB cell extract followed by HiTrap Heparin chromatography. The histidine tag was removed from purified TtSmtB by thrombin digestion (Sigma). The purified protein was stored in aliquots at − 20 °C [24 (link)].