Chromatography, Agarose

Expression plasmid DNA was transformed into E. coli BL21-Tuner cells. Single colonies were grown in 500 ml of LB to OD₆₀₀ ∼0.6 and fusion proteins induced by addition of 300 μM IPTG. Cells were grown at 37 °C prior to induction and then shifted to 30 °C for 4 h before harvesting by centrifugation at 10,800 g for 5 min. Cells were pelleted and stored at −80 °C until needed. Native purifications of His₆-tagged proteins were performed using affinity chromatography with Ni-NTA agarose beads (Qiagen). All steps of the purification (except for elution) were performed in batch using 50-ml conical tubes. The following buffers were used for purification: lysis buffer (20 mM Tris-HCl [pH 7.9], 0.5 M NaCl, 10% glycerol, 20 mM imidazole, 0.1% Triton X-100, 1 mM PMSF, 1 mg/ml lysozyme, 125 units benzonase nuclease [Novagen]), wash buffer (20 mM HEPES-KOH [pH 8.0], 0.5 M NaCl, 10% glycerol, 20 mM imidazole, 0.1% Triton X-100, 1 mM PMSF), elution buffer (20 mM HEPES-KOH [pH 8.0], 0.5 M NaCl, 10% glycerol, 250 mM imidazole), and storage buffer (10 mM HEPES-KOH [pH 8.0], 50 mM KCl, 10% glycerol, 0.1 mM EDTA, 1 mM DTT). Each cell pellet was resuspended in 10 ml of lysis buffer, incubated at room temperature for 20 min, sonicated, and then centrifuged for 60 min at 30,000 g to generate a cleared lysate. His₆-tagged proteins were bound to 1 ml of Ni-NTA agarose slurry, washed twice with 50 ml of wash buffer, and then loaded onto an Econo-column (Bio-Rad, Hercules, California, United States) for elution. Purified protein was eluted using 2.5 ml of elution buffer and loaded directly onto a PD-10 column (Amersham Biosciences, Piscataway, New Jersey, United States) that had been pre-equilibrated with storage buffer. If necessary, samples were filtered with a 0.2-μm HT Tuffryn filter (Pall Gelman Sciences, East Hills, New York, United States), and then concentrated to approximately 1–10 mg/ml using Centricon YM-10 or YM-30 columns (Millipore, Billerica, Massachusetts, United States). All samples were filtered through an Ultrafree-MC (0.22 μm) spin filter (Millipore) and then aliquoted for storage at −80 °C. Protein concentrations were measured using Coomassie Plus Protein Assay Reagent and a BSA standard (Pierce Biotechnology, Rockford, Illinois, United States). An equal amount (500 ng) of each protein sample was analyzed by 12% SDS-PAGE to verify molecular weight and purity. Prior to phosphotransfer profiling, all response regulator concentrations were normalized against a 500-ng BSA standard using a ChemiImager 5500 and densitometry (Alpha Innotech, San Leandro, California, United States) (see Figure S5).

The crude protease was purified under cooling at 4 °C through four stages comprising (NH₄)₂SO₄ salt precipitation, Phenyl-Sepharose 6FF hydrophobic chromatography, DEAE-Sepharose CL-6B anion exchange chromatography, and Sephadex G-75 gel permeation chromatography. Initially, the culture broth was centrifuged at 7000×g for 10 min, then the addition of (NH₄)₂SO₄ at 30–70% saturation was performed. Centrifugation at a speed of 10,000×g for 15 min to harvest the soluble proteins. Pelleted crude protease and other proteins were then resuspended at pH 7.8 in buffer A (20 mM Tris–HCl comprising 1.0 M )NH₄)₂SO₄. After the elimination of the suspended matter and the dialysis step, the concentrated crude protease was loaded into a Phenyl Sepharose 6 FF column of 1.5 × 20 cm² dimension. Thereafter, a linear ascent of 0.5–0.0 M (NH₄)₂SO₄ in buffer A was applied for the elution of proteins. From this elution chromatogram, active fractions showing enzymatic activity were pooled and concentrated. Thence, loaded onto the next column comprising of DEAE-Sepharose CL-6B with 1.5 × 15 cm² dimension. Elution was done at a 0.5 mL/min flow rate at pH 9.4 with 20 mM Tris–HCl buffer (buffer B). Active fractions displaying proteolytic activity were then concentrated, dialyzed with buffer B, and loaded into the next column comprising Sephadex G-75 FF with a 1.5 × 30 cm² dimension. Elution was done by buffer A. Final protease-active fractions were lyophilized and SDS-PAGE was performed by the universal technique of Laemmli^{11 (link)} using 15% (w/v) separating gel and 5% (w/v) stacking gel.

The coding sequences of the selected candidate genes, OePPO1 (OE6A068152), OePPO2 (OE6A114203), OePPO3 (OE6A046766), and OePPO4 (OE6A110596), were synthesized lacking the chloroplast transit peptide (amino acids 1–92, 1–93, 1–84 and 1–98, respectively) with the Escherichia coli codon optimization (GenScript, Piscataway, NJ, USA) and cloned into a pGEX6P.1 vector as EcoRI-XhoI (OePPO1-3) or BamHI-SalI (OePPO4) fragments. Four constructs were obtained to be expressed as glutathione-S-transferase (GST) fusion proteins: GST-OePPO1 to GST-OePPO4. Protein expression and purification were carried out according to Kampatsikas et al. [13 (link)] with minor modifications for each OePPO construct. BL21(DE3) lacIq E. coli cells containing the corresponding OePPO construct were grown at 37 °C in Luria–Bertani media (LB) with NaCl 0.5 M to OD₆₀₀ of 0.6, then supplemented with 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) and 0.5 mM CuSO₄ and grown for 20 h at 19 °C. Cells were harvested by centrifugation, suspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 1 mM EDTA, 10% glycerol, 1 mM benzamidine, and 1 mM PMSF), and lysed by sonication. The resulting crude protein lysate was clarified by centrifugation prior to chromatographic purification with sepharose-GSH beads (GE Healthcare, Chicago, IL, USA) following the manufacturer’s instructions. The soluble fraction obtained was added to the beads for GST-OePPO purification and incubated for 2 h at 4 °C in a 360° rotator. Then, recombinant protein-bound beads were incubated in a preScission Protease proteolytic digestion buffer (GE Healthcare) (50 mM Tris HCl pH 7.0, 150 mM NaCl, 1 mM EDTA) with 160 U/mL of protease. The enzymatic digestion was carried out overnight at 4 °C on a 360° rotator to eliminate the GST fusion protein from the OePPO proteins. The purified OePPO proteins were collected as eluates after centrifugation. The purified protein buffer was exchanged to 100 mM Tris-maleate, pH 6.8, and 10% glycerol buffer on a PD-10 column (Sephadex G-25, GE Healthcare, Chicago, IL, USA) and further concentrated using a Vivaspin centrifugal concentrator (MWCO 30 kDa, Merck, Darmstadt, Germany). The purity of each of the recombinant proteins was evaluated by SDS-PAGE, and the protein concentration was determined by the Bradford assay [29 (link)]. The control protein extract consisted of untransformed E. coli BL21 cells grown and subjected to the same purification steps.

E6006 bacteria were grown at 30 °C in M63 medium [46 ] with 10 mM glucose to an OD₆₀₀ of 0.2. The cells were then heated to 45 °C by adding medium that had been pre-warmed to 60 °C. The cultures were incubated for 1 h, then cooled to 4 °C on ice. Cells were collected by centrifugation at 8000× g for 10 min, with the resulting pellets re-suspended in buffer A (50 mM Tris-HCl pH 7.5, 15 mM Mg(C₂H₃O₂)₂, 100 mM NH₄Cl, 3 mM HOCH₂CH₂SH, 0.5 mM EDTA, and 1 mM PMSF) and disrupted using a French press (1250 PSI). The lysate was centrifuged at 15,000× g for 30 min at 4 °C. The supernatant (250 A₂₆₀ units) was centrifuged on a 38-mL (10–40%) sucrose gradient using an SW 28 rotor working at 21,000 rpm for 17 h at 4 °C. Fractions of interest were then pooled from different gradient tubes and concentrated via centrifugation at 100,000× g for 6 h. Pellets were dissolved in buffer A then analyzed using IMAC chromatography with Ni-NTA resin (Qiagen, France). All 70S ribosomes and 30S fractions containing his-tagged uS2 proteins were eliminated by three successive Ni-NTA purification steps. Particles retained by the resin were eluted with 100 mM imidazole in buffer A. The effluent underwent chromatography on Strep-Tactin sepharose (IBA, Göttingen, Germany) to bind particles containing the bS20 strep protein. These particles were eluted with 2.5 mM desthiobiotin in buffer A, concentrated with Amicon (100MCW) cartridges, and then analyzed by SDS-PAGE electrophoresis. The purification of mature 30S subunits from E6006 cells was done in the same way as described here for the precursors.