Histrap ff 5 ml nickel affinity column

The expression and purification of CHIKV-nsP2-CT was according to the previously described procedure (54 (link)) with some modifications. In brief, the transformed Rosetta 2 (DE3) cells were grown in Luria Bertani broth (HiMedia, India). The overexpression was induced with 0.4 mM isopropyl-d-1-thiogalactopyranoside (Sigma, MA) and allowed to proceed for 16 h at 18°C. The cell pellets after overexpression were collected by centrifugation and resuspended in lysis buffer having 50 mM Tris (pH 7.5), 500 mM NaCl, and 10 mM imidazole. The cells were disrupted by sonication using a Vibra-Cell probe-type ultrasonic processor (Sonics, CT). The lysed sample was clarified by centrifugation. The supernatant containing the soluble protein was passed through a pre-equilibrated HisTrap FF 5-mL nickel affinity column (Cytiva, MA). The column wash step was performed using a buffer containing 50 mM Tris (pH 7.5), 250 mM NaCl, 10 mM imidazole, and 1 mM phenylmethylsulfonyl fluoride (PMSF), and the bound protein was eluted with gradient against a buffer containing 50 mM Tris (pH 7.5), 250 mM NaCl, 250 mM imidazole, and 1 mM PMSF. The protein was then subjected to size exclusion chromatography using a HiLoad 16/600 Superdex 200-pg column (Cytiva) in a buffer containing 25 mM Tris (pH 7.5), 100 mM NaCl, 5%, glycerol, and 0.5 mM DTT. The peak fraction was analyzed by SDS-PAGE.