Sephadex g25 beads

The pDEST-HisMBP vector carrying TMV MP and pET-28a vector carrying MBP were transfected into E. coli strain BL21 (EMD Millipore). Bacteria were cultivated at 37°C until a A600 value close to 0.6 was reached. IPTG was added to 0.1 mM and, following incubation for a further 4 h, cultures were centrifuged at 5000 g for 15 min at 4°C. To extract protein, cell pellets were resuspended in native binding buffer (50 mM NaH₂PO₄, 0.5 M NaCl, pH 8.0, 0.1% (v/v) Triton X-100). Lysozyme (1 mg/ml) was then added to the cell suspension. Cell lysis was performed on ice for 30 min. After sonication, cell lysates were centrifuged at 5000 g for 15 min at 4°C to collect supernatant, which was incubated with Ni-NTA agarose (Qiagen) to adsorb His tagged fusion protein. Ni-NTA agarose and bound protein were collected by centrifugation. After washing the pellet with native washing buffer (50 mM NaH₂PO₄, pH 8.0, 0.5 M NaCl, 20 mM imidazole), fusion protein was eluted using native elution buffer (50 mM NaH₂PO₄, pH 8.0, 0.5 M NaCl, 0.25 M imidazole). Gel filtration was performed with Sephadex G-25 beads (Sigma-Aldrich) prior to subsequent assays. Proteins were visualized by Coomassie blue staining of a 10% SDS-polyacrylamide (37.5:1) gel.