Fig. 1
Analysis of TEV CP proteins expression in E. coli lysates and purification. The cells were cultured in Terrific Broth medium (Sigma-Aldrich, USA) and grown at 30 °C, until OD600 reached 1.0. Overnight induction was performed at 20 °C. 1 mM IPTG was used as inducer and cells were lysed by sonication in 20 mM Tris, pH 8.0 (matrix buffer, MB), 500 mM NaCl. a Immune detection analysis of TEV CP proteins using anti-6xHisTag antibody (Roche, Switzerland) for western blot (WB) and anti-TEV antibody (Agdia, USA) for ELISA. b Analysis of the purification process of His-TEV-CP by 12% SDS-PAGE loading 10 μl of sample per lane. The purification process was performed using His Trap HP IMAC 1 ml columns (GE-Healthcare, USA). Total soluble protein (TSP) in MB, 500 mM NaCl and 10 mM imidazole was loaded into the column. The flow-through (FT) was collected. Washing steps were performed in MB, 40 mM imidazol, and NaCl gradient (adjusted every 5 ml at 200, 400, 600 and 800 mM). The wash FT (WFT) was collected in 4 fractions, respectively. The proteins were eluted using MB, 100 mM NaCl and 500 mM imidazol. The elution FT (EFT) was collected in 6 fractions of 1 ml each. c Analysis of purified His-TEV-CP by 12% SDS-PAGE and WB using anti-His antibody loading 10 μl of sample per lane. The first two fractions of EFT were prepared in MB, 500 mM NaCl, using 3 kDa Amicon Ultra centrifugal filter devices (Merck-Millipore, Germany). Protein concentration was estimated against a BSA curve using Bradford reagent