Source 15q column

E. coli BL21 (DE3) was transformed with plasmid DNA encoding maltose-binding protein fused to pX with an intervening tobacco etch virus (TEV) cleavage sequence, and grown at 37°C until OD600 = 1.0. Cells were induced with 1 mM IPTG and allowed to grow overnight at 37°C. Cells were harvested by centrifugation and the pellet resuspended in buffer (20mM Tris pH 7.4, 200 mM NaCl, 1 mM EDTA, 1mM DTT, cOmplete Protease Inhibitor cocktail (Roche)). A French press was used to lyse cells and the cell debris pelleted by centrifugation at 15,000 rpm. Supernatant was passed over an amylose resin column (New England Biolabs) and the column washed with 10 column volumes of buffer (20mM Tris pH 7.4, 200 mM NaCl, 1 mM EDTA). Protein bound to the amylose column was eluted using elution buffer (20mM Tris pH 7.4, 200 mM NaCl, 1 mM EDTA, 10 mM maltose). The eluate from the amylose column purification was digested with TEV protease and subjected to anion exchange chromatography (SOURCE 15Q column, GE Healthcare) and bound proteins eluted with an increasing NaCl gradient. Fractions were analyzed by SDS-PAGE and fractions containing pX protein were pooled and concentrated using a Vivaspin 20 concentrator (3000 MWCO, Sartorius). The final protein preparation was visualized on SDS PAGE and concentration determined using the bicinchoninic acid (BCA) protein assay.

Frozen M. extorquens AM1 cells with pCM110-MeFDH1 were lysed in buffer A (50 mM Tris–HCl, pH 8.0) with 1 mM PMSF by passing them through a microfluidizer and centrifuged at 4611 × g (Vision V506CA rotor) for 30 min at 4 °C to remove the cell debris. The supernatant was applied to a Ni–NTA affinity column (Qiagen) equilibrated with buffer (50 mM Tris–HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole). The protein was eluted with a linear gradient of 100–500 mM imidazole. Protein fractions were diluted two-fold in buffer A and loaded onto a Source 15Q column (GE Healthcare) equilibrated with buffer A. The protein was eluted with a gradient of 0–600 mM NaCl, and MeFDH1 was eluted at about 300 mM NaCl. Further purification used size exclusion chromatography (HiLoad 16/600 Superdex 200 prep grade, GE Healthcare) on a column that was previously equilibrated with a buffer containing 50 mM Tris–HCl pH 8.0 and 100 mM NaCl. Anti-His-tag antibodies were used for western blot analysis to confirm the presence of the His tag and the C-terminal cap domain.

The periplasmic protein fraction was isolated using osmotic shock. In brief, the cells were thawed on ice, resuspended in a buffer containing 20% sucrose, 1 mm EDTA and 10 mm Tris/HCL, pH 8.0, and incubated on ice for 15 min. The resulting spheroplasts were removed by centrifugation at 10 000 g at 4 °C for 15 min. The supernatant was dialyzed against 10 mm Tris/HCL (pH 8.0) and used for further purification. Ion exchange chromatography was performed on a SOURCE™ 15Q column (10 cm × 0.75 cm², GE Healthcare Life Sciences, Freiburg, Germany), equilibrated with the same buffer; the recombinant enzyme preparation was eluted with a linear gradient (0–300 mm NaCL, 2 mL·min⁻¹). The estimated purity of all the recombinant proteins was of more than 95%. Fractions containing the active enzyme were stored at +4 °C or frozen at −20 °C.

Nucleosomes were assembled using DNA fragments derived from the MMTV-A positioning sequence (Flaus and Richmond, 1998 (link); Flaus and Owen-Hughes, 2003 (link); Ferreira et al., 2007 (link)), and were purified as unmodified or acetylated H3/H4 tetramers and a stoichiometric amount of H2A/H2B dimers. All DNA fragments were made by PCR using fluorescently labelled primers. The PCR products were purified by anion exchange chromatography on a 1.8 ml SOURCE 15Q column (GE Healthcare Life Sciences) or native (0.5× TBE) PAGE. The notation xAy denotes the 147-bp MMTV-A sequence with flanking DNA of x and y bp on the upstream and downstream side, respectively. Typically, assembly was performed by salt-gradient dialysis using a double-dialysis method (unless specified otherwise), as follows—reactions (25–35 µl) were placed in microdialysis buttons, which were placed inside a dialysis bag containing 30 ml 0.5× TE and 2 M NaCl, the dialysis bag was then dialysed overnight against 2 L of 0.5× TE at 4°C.

The SARS-CoV-2 3CLpro proteins were overexpressed in Escherichia coli and purified following a method described previously^{21 (link)}. In brief, the His-tagged proteins were purified by affinity chromatography using Co²⁺ resin (TALON, 635504), then the tag was removed by human rhinovirus 3C protease (TaKaRa, 7360) and the efficiency of cleavage was analyzed by SDS-PAGE (GenScript, M00656). The proteins were further purified by ion-exchange chromatography (Source-15Q column, GE Healthcare) and size-exclusion chromatography (Superdex 200 increase 10/300 GL column, GE Healthcare). Finally, the purified proteins were concentrated and stored in 20 mM HEPES, pH 7.4, 150 mM NaCl at –80 °C for subsequent biological assays and crystallization.

To create WRC:Abi2-(MBP)₂, the sortase ligation sequence, LPGTG, was genetically fused to the C-terminus of MBP-Abi2 (1-158). Meanwhile, a TEV site was added to the N-terminus of an (MBP)₂ tag, which after Tev cleavage would expose a Gly required for sortase ligation. MBP-Abi2 (1-158)-LPGTG was expressed, purified, and incorporated into the WRC as described above to create WRC-LPGTG. GG-2MBP was expressed in Arctic Express (DE3) RIL (Agilent) cells after induction with 0.75 mM IPTG at 10°C for 16 hr, purified on amylose resin, and subjected to TEV cleavage overnight, followed by anion exchange chromatography using a Source 15Q column (GE Healthcare). Sortase5M (sortase A pentamutant) was a gift from David Liu (Addgene plasmid # 75144), expressed in Arctic Express (DE3) RIL (Agilent) cells, purified over Ni-NTA agarose resin, followed by cation exchange using a Source 15S column (GE Healthcare) and size exclusion chromatography using a Superdex 75 column (GE Healthcare) (Chen et al., 2011 (link)). WRC-LPGTG (1 µM) was mixed with GG-MBP (25 µM) and sortase (10 µM) in 50 mM Tris pH 7.5, 150 mM NaCl, and 10 mM CaCl₂ and left at RT for 2 hr. The reaction was quenched by adding 25 mM EGTA, and the WRC-(MBP)₂ was purified over a Superdex 200 column to separate the WRC-(MBP)₂ from unligated molecules and sortase.