Probond nickel chelating resin

Recombinant human ERCC1–XPF wild-type protein (containing polyhistidine (His-6) tags) was expressed from a bicistronic plasmid (kindly provided by Dr. Richard Wood, University of Texas MD Anderson Cancer Center, Smithville, TX) in the E. coli BL21(DE3) strain, Following previously described procedures (70 (link), 78 (link)) the proteins extracted from E. coli were eluted from a ProBond Nickel-Chelating Resin (Thermo Fisher Scientific) and then a Hi-trap heparin column (GE Healthcare). Fractions containing ERCC1–XPF were dialyzed, concentrated, and stored at −80°C in 10 mM HEPES, pH 7.4, 2.5 mM β-mercaptoethanol, 0.01% CHAPS, 0.25 mM EDTA, 50% glycerol, and 25 mM NaCl. Based on polyacrylamide gel separation and Coomassie Blue staining, the final purity of the full-length ERCC1-XPF heterodimer was determined to be ~35%.

For transient expression of the protein, Freestyle 293-F cells (Thermo Fisher Scientific #R79007) were transfected with pSecTag2A plasmid according to the supplier’s protocol. After 4 days of culture, cells were pelleted by centrifugation at 300 × g for 5 min, and supernatant protein expression was confirmed by Coomassie gel stain (Thermo Fisher Scientific #24592) and western blotting with anti-Myc antibody (Abcam #ab62928). Proteins derived from transient transfection were purified as follows. Supernatant was passed through columns containing ProBond nickel chelating resin (Thermo Fisher Scientific #R80101). Then, each column was washed four times with native purification buffer (50 mM NaH2PO4 and 0.5 M NaCl pH 8.0) plus 20 mM imidazole (Sigma–Aldrich #I5513), and then eluted three times with native purification buffer plus 250 mM imidazole concentrations. Eluted proteins were concentrated to ~2 mL and dialyzed into 1× PBS (Thermo Fisher Scientific #AM9625). After dialysis, the protein was verified by western blot and SDS-PAGE gel electrophoresis and protein concentration was quantified by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific #23227).

Recombinant protein expression and purification was carried out as previously described (10 (link)). Briefly, E. coli BL21 (DE3) transformed with the plasmid for GLuc or EC1-GLuc was grown in a LB medium containing 100 μg/ml ampicillin at 37°C. When the OD₆₀₀ reached 0.6, the expression of the recombinant proteins was induced by the addition of 0.2 mM isopropyl 1-thio-β-D-galactopyranoside at 25°C overnight. The GLuc-6His and EC1-GLuc-6His proteins were purified from the supernatant using ProBond Nickel-chelating resin (Invitrogen). The recombinant proteins were subjected to 15% SDS-PAGE, and the expression was confirmed by Coomassie brilliant blue staining and western blot analysis with rabbit anti-GLuc serum (1:1,000; Nanolight Technology, Pinetop, AZ, USA). The proteins were finally dialyzed against PBS (pH 7.4) at 4°C for 24 h, and the concentrations were determined using a Biadford protein assay kit (Bio-Rad). Aliquots of protein were stored at −80°C prior to use.

Total protein from leaves of sweet potato was extracted with extraction buffer [20mM Tris/HCl (pH 7.2) and 1% Protease Inhibitor Cocktail]. The extracted protein was incubated with reaction mixture containing 20mM Tris/HCl (pH 7.2), 10mM MgCl₂, 0.5mM CaCl₂, 0.5mM ATP, 2mM dithiothreitol, and 10 μg of purified IbMAPK–His. After incubation at 30 °C for 1h, ProBond™ Nickel-Chelating Resin (Invitrogen) was added to purify IbMAPK–His. After washing five times, the bound proteins were eluted and separated by 12% SDS-PAGE, and detected by immunoblotting using an anti-pERK andtibody (Santa Cruz) and an anti-His antibody (LTK Biolaboratories).

A second purification method was performed on the protease enzyme through Probond Nickel Chelating Resin (Invitrogen) affinity chromatography. After ammonium sulfate precipitation had been performed, the dialyzed sample was connected to Probond affinity column equalized with a natural-binding buffer (0.5 M NaCl and 50 mM NaH₂PO₄, pH 8.0). After the removal of the proteins which did not contain histidine amino acid by using a wash buffer (0.5 M NaCl, 20 mM imidazole, and 50 mM NaH₂PO₄, pH 8.0), the protease enzyme was eluated from the column by using an elution buffer (0.5 M NaCl, 250 mM imidazole, and 50 mM NaH₂PO₄, pH 8.0). Enzyme and protein assays were performed on the collected fractions.