Ni nta sepharose column

E. coli BL21(DE3) (Agilent Technologies) carrying pGGE6 derivatives were grown in TB (terrific broth) medium [45 ]. Expression was induced at an OD₆₀₀ of 0.6 to 0.9 in presence of 0.5 μM IPTG for 18 h at 16°C. Cells were harvested by centrifugation, resuspended in ice-cold lysis buffer (50 mM Tris/HCl, 10 mM NaCl, 10 mM imidazol, 0.1% Tween20, pH 8.0), supplemented with protease inhibitors (Roche), and lysed by three freeze-thaw cycles. Protein extracts were separated by Ni-NTA sepharose column (QIAGEN), equilibrated with lysis buffer. After washing with lysis and wash buffer (50 mM Tris, 10 mM NaCl, 40 mM imidazol, 0.1% Tween20, pH 8.0), respectively, His₆-tagged proteins were eluted with elution buffer (50 mM Tris, 10 mM NaCl, 250 mM imidazol, 0.1% Tween20, pH 8.0) at 4°C.
For protein purification under reducing conditions, buffers were supplemented with 1 mM Tris (2-carboxyethyl) phosphine (TCEP). Elution fractions containing enriched protein were pooled and dialysed 2× 5 h with 200× volumes of storage buffer (50 mM Tris, 10 mM NaCl, 10% glycerol, pH 8.0). Protein concentrations were determined at 280 nm using the molar absorption coefficient calculated according to Pace et al. [46 (link)], or by Bradford assay (BioRad, Hercules, USA). Protein aliquots were frozen in liquid nitrogen and stored at -80°C.

E. coli C41 (DE3) pLysS was obtained from Novagen. pET28a expression vector was purchased from Invitrogen. Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Medium F12 (DMEM F12) were purchased from Gibco, plasmid extraction kit and Ni-NTA-Sepharose column from Qiagen. IPTG obtained from Vivantis. Luciferin was purchased from Resem (the Netherlands). Adenosine triphosphate (ATP) and dialysis bag (2.5 KDa) were purchased from Sigma. MTT 3-(4,5-s-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide was obtained from Sigma–Aldrich. Graphite powder (mesh~200 µm), KMnO₄, NaNO₃, H₂O₂, Dimethylformamide (DMF) were purchased from Chemlab. Sulfuric acid (98%) was obtained from Merck.

The sequences of selected HPV16 E7-binding affibodies molecules, including Z_{HPV16 E7}127, Z_{HPV16 E7}301, Z_{HPV16 E7}384, Z_{HPV16 E7}745 and wild SPA-Z scaffold (Z_-WT), were subcloned into the Nde I and EcoRI sites of pET21a(+) expression vector. Following confirmation of the inserted sequences by enzyme digestion and DNA sequencing, positive plasmids were transformed into E. coli BL21 (DE3) for expression of the fusion proteins. The 6×His-tagged recombinant proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by Western blotting using anti-His antibody (Sigma). The recombinant protein was purified by affinity chromatography using precharged Ni-NTA Sepharose column (Qiagen) and refolded when dialyzed in PBS using Slide-A-Lyzer (Pierce) according to the manufacturer's recommendations. The purity of purified proteins was verified by SDS-PAGE, and the protein concentrations were determined by the bicinchoninic acid (BCA) protein quantitation method.