Ni nta bead

All recombinant proteins were expressed in Escherichia coli BL21(DE3) CodonPlus cells (Stratagene) in LB (for crystallographic studies) or M9 minimal medium (for NMR studies) containing ¹³C-glucose and/or ¹⁵NH₄Cl as sole sources of carbon and nitrogen, respectively. All recombinant proteins used for structural and biophysical characterization were lysed by sonification in lysis buffer (50 mM sodium phosphate (pH 7.5), 300 mM NaCl, 10 mM imidazole, and 1 mM DTT). Cell debris was removed by centrifugation and the filtered supernatant applied to Ni-NTA beads (Quiagen) for affinity chromatography. Purified proteins were eluted with lysis buffer containing 300 mM imidazole. The elution fractions were supplemented with His-tagged TEV protease (except for the RPN10 UIM2) and dialyzed in dialysis buffer (50 mM sodium phosphate (pH 7.5), 300 mM NaCl, and 1 mM DTT). After cleavage, the desired proteins were separated from His₆-tagged cleavage products and His₆-tagged TEV protease by Ni-affinity chromatography using dialysis buffer. All proteins were further purified by size-exclusion gel filtration. Pure proteins for NMR and DSF analyses were buffer exchanged to NMR buffer (20 mM sodium phosphate pH 7.5, 150 mM NaCl) that contained 1 mM DTT for proteins containing Cys residues. For crystallization, proteins were in 20 mM HEPES (pH 7.5), 150 mM NaCl.

All histones were bacterially expressed as His₆-tagged recombinant proteins^{12 (link),58 (link)–60 (link)}. Human histones H2A, H2B, H3.1, H3.1^CATD, and H3.1^{CATD(V76Q, K77D)} were produced in E. coli BL21(DE3). CENP-A, CENP-A^QD was produced in E. coli BL21-CodonPlus(DE3)-RIL. Human histone H4 was produced in E. coli JM109(DE3). The Se-Met-substituted H2A ^{L51M, L58M, L93M} and Se-Met-substituted H2B were produced in E. coli B834(DE3). Expressed histones were corrected from the inclusion body and purified by Ni affinity chromatography using Ni-NTA beads (QIAGEN) under denaturing condition. His₆-tags of these purified histones were removed by treatment with thrombin protease (Wako or GE Healthcare) under non-denaturing condition. After thrombin protease treatment, histones were further purified by ion exchange chromatography using MonoS column (GE Healthcare) under denaturing condition. Purified histones were dialyzed against water containing 2 mM 2-mercaptoethanol and lyophilized. Lyophilized histone powders were stored at 4 °C.

T7 lysozyme was expressed as C-terminally His-tagged fusion protein in BL21 DE3 RIL cells; 1 L of cells was grown to log phase, induced with 1 mM IPTG at 37°C for 3 hr. Cells were harvested by centrifugation for 30 min at 4000 rpm and resuspended in buffer L (50 mM Tris-HCl pH 7.6/10% glycerol/0.02% NP-40)/300 mM NaCl/protease inhibitor cocktail/1 mM DTT. Cells were lysed by incubation with 0.1 mg/mL lysozyme for 30 min on ice followed by sonication. Cleared lysate was obtained by centrifugation at 15,000 rpm in an SS34 rotor for 30 min at 4°C. The clarified extract was supplemented with 10 mM imidazole and rotated for 2 hr at 4°C with 1 mL of Ni-NTA beads (Qiagen). Bound protein was eluted with 10 CV of buffer L/200 mM imidazole. Peak fractions were pooled, diluted with buffer L to lower the salt concentration to 10 mM NaCl, and fractionated over a 1 mL MonoS column using a 20 CV gradient of 10–125 mM NaCl in buffer L. Peak fractions were pooled, concentrated using Amicon Ultra 4 mL centrifugal filters (Millipore) and fractionated over 24 mL S200 gel filtration column equilibrated in buffer L/125 mM NaCl. Peak fractions were pooled, aliquoted, flash-frozen, and stored at –80°C.

The recombinant proteins were expressed in Lenti-X 293T cells by calcium phosphate transfection and purified by affinity chromatography with protein A (GE Healthcare) and Ni-NTA beads (Qiagen). Gel filtration of the proteins were performed on an ÄKTA purifier system with Unicorn 5.1 software (GE Healthcare) using PBS as running buffer at a constant flow rate of 0.1 ml/min. 200 μg protein was loaded on a Superdex 200 10/300 GL column (GE Healthcare). Ovalbumin and aldolase of the Gel Filtration HMW Calibration Kit (GE Healthcare) were used as molecular weight controls. Quantifications were done by capillary electrophoresis (Experion Pro260 kit; Bio-Rad). Purity and molecular masses of TP15-Fc and 4D5-Fc (5 μg protein each) were analyzed on Coomassie gel by using standard procedures.