Talon cobalt column

Construct expression of the NTS-DBL1α-domain was performed as previously described [24 (link)]. In brief, the NTS-DBL1α-domain spanning amino acids 1–481 (PFIT_bin06900) was codon optimized for expression in bacteria and cloned into the pJ414express vector (DNA2.0). The protein was expressed as a C-terminal 6× histidine-tagged recombinant protein in Escherichia coli (Shuffle T7, New England Biolabs); bacteria were grown at 30 °C until they reached an OD₆₀₀ = 0.6–0.8 and then induced with 0.4 mM IPTG (isopropyl-β-D-thiogalactopyranoside) for 20 h at 16 °C. Pelleted bacteria were lysed by sonication and the recombinant protein was recovered from the soluble fraction after centrifugation. The protein was purified by immobilized metal affinity chromatography (IMAC) over a TALON Cobalt column (Clontech), followed by size exclusion chromatography on a HiLoad 16/60 Superdex 75 pg column (GE-Healthcare). The purified protein was analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot using an antibody against the poly-His tag (Qiagen).

Bacteria with the plasmids encoding the desired gene were grown to OD600~0.6 at 37 °C, and 1 mM IPTG was added to induce protein expression. After shaking for 4 hours, cells were harvested by centrifugation and re-suspended in a lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100, 0.4 mM PMSF and 2 mM β-mercaptoethanol, pH 7.5). For the purification of OmpR, bacteria were lysed by sonication and protein was purified using a TALON Cobalt column (Clontech).
High salt wash buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, 2 mM β-mercaptoethanol, pH 7.5) and low salt wash buffer (20 mM Tris-HCl, 150 mM NaCl, 20 mM imidazole and 2 mM β-mercaptoethanol, pH 7.5) were used to eliminate non-specific binding. The OmpR protein was eluted by elution buffer (20 mM Tris-HCl, 150 mM NaCl, 250 mM imidazole and 2 mM β-mercaptoethanol, pH 7.5).
For purification of EnvZ, supernatant was loaded on the glutathione column (GE healthcare), washed with buffer (20 mM Tris-HCl, 350 mM NaCl and 2 mM β-mercaptoethanol, pH 7.5) and eluted by elution buffer (20 mM Tris-HCl, 150 mM NaCl, 10 mM reduced glutathione and 2 mM β-mercaptoethanol, pH 7.5). All purified proteins were dialyzed against storage buffer (20 mM Tris-HCl, 200 mM NaCl and 10% glycerol, pH 7.5) at 4 °C overnight. Protein purity and concentration were determined by A280/A260 and SDS-PAGE (Purity > 95%).

The open-reading frame of E. coli hfq was amplified from MG1655 and cloned into pET28a to generate pET28a-hfq with a C-terminal 6 × His tag, and this plasmid was transformed into E. coli BL21(DE3)pLysS for protein purification. Cells containing pET28a-hfq were grown to OD₆₀₀ ~0.6 at 37°C, and Hfq protein was induced by adding 2 mM IPTG. After shaking for 4 h, cells were harvested by centrifugation and resuspended in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100, 0.4 mM PMSF, and 2 mM ß-mercaptoethanol, pH 7.5). Then, bacteria were lysed by sonication and precipitate was removed by centrifugation. Supernatant was passed over TALON Cobalt column (Clontech), followed by washing with five bed volumes of high salt buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, 2 mM ß-mercaptoethanol, pH 7.5) and low salt buffer (20 mM Tris-HCl, 150 mM NaCl, 20 mM imidazole and 2 mM ß-mercaptoethanol, pH 7.5). Hfq protein was eluted by elution buffer (20 mM Tris-HCl, 150 mM NaCl, 250 mM imidazole, and 2 mM ß-mercaptoethanol, pH 7.5). Purified Hfq was dialyzed against storage buffer (20 mM Tris-HCl, 200 mM NaCl and 10% glycerol, pH 7.5) at 4°C overnight. Hfq purity and concentration were determined by A₂₈₀/A₂₆₀ and SDS-PAGE (purity > 95%).