Ni2 nta agarose affinity column

Escherichia coli harboring pCra-His₆ was cultivated in LB broth and treated with 0.2% arabinose for 3 h for Cra-His₆ induction (81 (link)). Bacterial cells were harvested at 10,000 × g for 10 min and treated with 1 mg/mL lysozyme in lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, and 20 mM imidazole, pH 8.0) on ice for 30 min. The cell suspension was sonicated and centrifuged at 10,000 × g at 4°C for 20 min. The soluble lysate fraction was then mixed with Ni²⁺-nitrilotriacetic acid (Ni²⁺-NTA) agarose beads (Qiagen) and incubated at 4°C for 1 h with gentle agitation. The reactant was loaded onto a Ni²⁺-NTA agarose affinity column (Qiagen). The column was washed with washing buffer (50 mM NaH₂PO₄, 300 mM NaCl, and 40 mM imidazole, pH 8.0) three times, and Cra-His₆ bound to the column was eluted using elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, and 300 mM imidazole, pH 8.0). The eluted fraction was placed in a dialysis membrane tubing with 10 K MWCO (SnakeSkin Dialysis tubing, Thermo Scientific Inc.) and incubated in dialysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1 mM EDTA, 5 mM DTT, and 20% glycerol) at 4°C. Protein concentration was measured using the Bradford assay (Bio-Rad) (82 (link)).

All the recombinant proteins were cloned, expressed and purified according the detailed procedure presented in SI Text. After Ni²⁺-NTA agarose affinity column (Qiagen), the proteins were further purified on a Superdex S200 10/300 GL or S200 16/600 column (GE Healthcare Inc.) using 25 mM Tris pH 8, 150 mM NaCl buffer. The proteins were analyzed by SDS-PAGE gel and stored at −80°C. The preparation of bulk and in vitro transcripts tRNAs are described in the SI Text. All the rationale and the procedure used to identify and delineate HsDus2 domains are described in the SI Text.

Escherichia coli strains producing His₆-tagged RpoS, RpoD, and Crl proteins were cultivated in LB medium broth, and the proteins were induced by adding 0.05mM (RpoS and RpoD) or 1mM (Crl) isopropyl β-D-1-thiogalactopyranoside for 7 or 3h at 30°C. Bacterial cells were centrifuged at 10,000×g for 10min and resuspended in lysis buffer (50mM NaH₂PO₄, 300mM NaCl, 20mM imidazole, and pH 8.0) containing 1mg/ml lysozyme. After 30min incubation on ice, the cells were sonicated and centrifuged at 10,000×g and 4°C for 20min. The resultant soluble lysate fraction was treated with Ni²⁺-nitrilotriacetic acid (Ni²⁺-NTA) agarose beads (Qiagen) at 4°C for 1h with rotation and loaded onto a Ni²⁺-NTA agarose affinity column (Qiagen). The column was washed with washing buffer (50mM NaH₂PO₄, 300mM NaCl, 40mM imidazole, and pH 8.0) three times, and the proteins were eluted with elution buffer (50mM NaH₂PO₄, 300mM NaCl, 300mM imidazole, and pH 8.0). The eluted protein fraction was packed into SnakeSkin™ Dialysis tubing with 10K MWCO (Thermo Scientific Inc.) and subjected to dialysis at 4°C in dialysis buffer (20mM Tris–HCl, pH 8.0, 150mM NaCl, 0.1mM EDTA, 5mM DTT, and 20% glycerol). Purified proteins were quantified using Bradford assay.