Ni-NTA Superflow (100 ml)

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Publication protocol

The purified pXT7 plasmids were transformed into E. coli BL21 Star (DE3) pLysS chemically competent cells according to the manufacturer’s protocol (Thermoscientific, USA). E. coli BL21 Star (DE3) pLysS cells containing 13 different plasmids were incubated with vigorous shaking at 37 °C up to an optical density (OD600) at of 0.4. Then, expression was induced with 0.5 mM IPTG (isopropyl-D-thiogalactopyranoside) for 4 h. Next, E. coli cells were harvested by centrifugation at 5000×g for 10 min. The cell pellets were homogenized with lysis buffer [0.1% Triton X-100, 50 mM Tris–Cl and 0.3 M NaCl (pH 7.4)] followed by 3 × freeze-thawing and then the processed samples were centrifuged at 30,000×g for 20 min at 4 °C. After centrifugation, the supernatants were incubated with 1 ml Ni–NTA Superflow beads (Qiagen, USA) shaking for 30 min. Next, the suspension was centrifuged at 2000×g for 1 min and the supernatant was discarded. Thereafter, the Ni–NTA beads containing recombinant proteins were washed with 50 mM Tris–Cl and 0.3 M NaCl and 25 mM imidazole (pH 7.4) for 30 min with shaking to remove the nonspecific bindings and centrifuged at 2000×g for 1 min. Ni–NTA beads containing recombinant proteins were incubated with 50 mM Tris–Cl and 0.3 M NaCl and 0.5 M imidazole (pH 7.4) for 30 min with shaking to release the proteins and centrifuged at 2000×g for 1 min. The molecular weight and level of recombinant protein expression in supernatants was determined by Western blotting as described below. The most abundantly expressed recombinant proteins were selected to be produced in larger amount and used as antigen in Rec-ELISA.

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