Ni nta agarose chromatography

Human histones (H2A, H2B, H3.1, CENP-A and H4) were expressed as N-terminally His₆-tagged proteins in Escherichia coli cells (47 (link)), and purified by nickel-nitrilotriacetic acid (Ni-NTA) agarose chromatography (Qiagen), thrombin protease (GE Healthcare) treatment, and Mono S column chromatography (GE Healthcare), as described previously (15 (link)). The human CENP-B DBD was expressed in E. coli cells, and was purified as described previously (46 (link)). H3.1^CATD was produced as a bacterially expressed recombinant protein, using the modified H3.1 expression vector. In the H3.1^CATD expression vector, the DNA fragment encoding amino acid residues 75–112 of H3.1 was replaced by the corresponding CENP-A region, encoding amino acid residues 75–114. H3.1^CATD was then purified by the same method as for H3.1 (48 (link)).

The DNA fragment encoding human histone H3.3 was inserted between the NdeI and BamHI sites of the pET21a-NHSP2 vector35 (link), in which the His₆-tag sequence, the Saccharomyces cerevisiae SUMO homolog, Smt3, and a PreScission protease cleavage sequence are located just upstream of the H3.3 coding sequence. Then, the N-terminally His₆-SUMO tagged H3.3 was expressed in Escherichia coli BL21(DE3) cells in the presence of isopropyl-β-D-thiogalactopyranoside (final concentration 1 mM). His₆-SUMO tagged H3.3 was recovered from inclusion bodies with 50 mM Tris-HCl (pH 8.0) buffer, containing 7 M guanidine hydrochloride, 500 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 5% glycerol, and was purified by nickel-nitrilotriacetic acid (Ni-NTA) agarose chromatography (Qiagen) under denaturing conditions with 6 M urea. His₆-SUMO tagged H3.3 was then purified by Mono S column chromatography (GE Healthcare) under denaturing conditions with 6 M urea. The purified His₆-SUMO tagged H3.3 was dialyzed against water containing 2 mM 2-mercaptoethanol, freeze-dried, and stored at 4°C.
Human histones H2A, H2B, H3.3, CENP-A, and H4 were expressed and purified as described previously26 (link)27 (link)36 (link).

MBP-tagged Fam20A and Fam20C proteins were expressed in Hi5 insect cells and purified from the conditioned medium as described previously (Xiao et al., 2013 (link)). The MBP tag was removed using gel filtration chromatography following TEV protease digestion. 6× His-tagged AMELX, AMTN, ENAM and SUMO-AMBN-N were expressed in E. coli BL21 (DE3) and purified by Ni-NTA-agarose chromatography (Qiagen, Venlo, Netherlands) as described previously (Tagliabracci et al., 2012 (link)). Flag-tagged Fam20C, Fam20B and decorin were expressed in 293T cells and purified from the conditioned medium as described previously (Wen et al., 2014 (link)).

The wild-type S. avermitilis genome was used as a template, and MtrA-F/MtrA-R were used as primers to amplify the mtrA gene. Linear fragments of the pET28a vector were obtained by amplification using pET28a-F/pET28a-R as primers. The expression vector pET28a-MtrA was constructed by seamlessly fusing the mtrA gene fragment with a linear fragment of the vector using the Seamless Cloning Kit. Subsequently, it was transformed into E. coli BL21 (DE3) to obtain the heterologous expression strain BL/pET28a-MtrA, which facilitates robust MtrA protein expression (Additional file 1: Fig. S4). Following induction by 0.2 mM IPTG, bacteria were collected, washed, resuspended in a lysis buffer, and sonicated on ice. Soluble MtrA protein was purified by Ni–NTA agarose chromatography (Qiagen) according to the manufacturer’s instructions. The purified protein was quantified by Quick Start Bradford Dye Reagent (Bio-Rad) and stored at − 80 °C.