Expression and purification of recombinant proteins in E. coli

To construct the vector coding for the histidine-tagged full-length NbHSP70 and TaHSP70 fusion proteins, NbHSP70 and TaHSP70 were amplified by PCR from cDNA libraries prepared from leaves of N. benthamiana or Triticum aestivum using primers pET-Nb70N/pET-Nb70C and pET-Ta70N/pET-Ta70C (Table S1), respectively. The amplified fragments were digested with BamHI/NotI and cloned into digested pET28a vector (Novagen). For construction of the vector coding for the C-terminus GST fusion of Rep^1–670 fusion protein, CWMV Rep^1–670 sequences were amplified from a CWMV cDNA library using the primer pair p6P1Rep^1–670 N / p6P1Rep^1–670 C (Table S1). The amplified fragments were digested with BamHI/EcoRI and cloned into digested pGEX-6P1 vector (GE Healthcare).

For purification of GST-tagged Rep^1–670, or the GST tag itself, a 20 ml overnight culture of E. coli BL21 cells containing the recombinant plasmid pGEX-Rep^1–670 or pGEX6P1 vector alone was used to inoculate 100 ml of LB media containing 75 μg/ml of carbenicillin. Cells were grown at 37 °C to an OD₆₀₀ of 0.4. Protein expression was induced with 0.1 mM of IPTG for 3 h for Rep^1–670-GST and GST. Bacterial cells were re-suspended in 20 ml of phosphate buffered saline solution (PBS; 4.3 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl, pH 7.3) supplemented with 1 mM DTT, 40 mg of lysozyme, and 1× complete EDTA-free protease inhibitor. The cells were disrupted by sonication and supplemented with 1% Triton-X-100. Lysate was centrifuged at 30,000 × g for 30 min at 4 °C. The supernatant was filtered through a 0.45 μm Millex HV PVDF filter (Millipore) and used for affinity purification of either Rep^1–670-GST or GST on glutathione sepharose 4B (GE Healthcare) according to the manufacturer’s protocol. For purification of histidine-tagged TaHSP70 and NbHSP70, a 5 ml overnight culture of E. coli BL21(DE3) cells containing the recombinant plasmid pET28(a)-TaHSP70 or pET28(a)-NbHSP70 was used to inoculate 100 ml of LB media containing 50 μg/ml of kanamycin. Cells were grown at 37 °C to an OD₆₀₀ of 0.4 to 0.6. Protein expression was induced with 1 mM of IPTG for 2 h at 37 °C. Bacterial cells were re-suspended in 5 ml of buffer A (50 mM sodium phosphate buffer pH 8.0, 300 mM NaCl, 5 mM β-mercaptoethanol, 0.1% Tween-20, 10% glycerol, 1× complete EDTA-free protease inhibitor). The cells were disrupted by sonication and the resulting lysate centrifuged at 30,000 × g for 30 min at 4 °C. The supernatant was used for immobilized metal affinity purification on Talon resin (BD Biosciences). Resin was washed with buffer A supplemented with 10 mM imidazole and proteins eluted with buffer B (20 mM Tris-HCl pH 7.5, 300 mM NaCl, 250 mM imidazole, 2.5 mM DTT). Concentration of all recombinant proteins produced was measured using a Bradford assay (Bio-Rad) using bovine serum albumin as standard. Protein purity and molecular weight were assessed by Coomassie staining and immunoblot analysis using monoclonal anti-histidine, monoclonal anti-GST.