P8112

6xHis-MBP-MISP and 6xHis-EGFP-MBP-MISP constructs were expressed in Sf9 insect cells. Insect cell pellets were resuspended in lysis buffer (20 mM Tris HCl, 0.3 M KCl, 10 mM imidazole, 10% glycerol, 2 mM DTT, pH 7.5) supplemented with protease inhibitors (Roche, 5892953001). Resuspended samples were lysed using a Dounce homogenizer and passed through an 18gauge needle to shear DNA. The resultant lysate was then centrifuged at 35,000 rpm in a Ti 50.2 rotor (Beckman) for 30 minutes at 4 °C. Clarified lysates were then filtered using a 0.45 µm syringe filter. Samples were then loaded into a HisTrap column according to the manufacturer protocol and eluted with a 50-500 mM linear imidazole gradient (pH 7.5). Protein purity was assessed by SDS-PAGE. Eluted protein was concentrated using a centrifugal filter (Millipore; UFC803024). For in vitro EM experiments, the 6xHis-MBP tag was cleaved from 6xHis-MBP-MISP using a TEV protease (NEB; P8112) for 1 hour at room temperature. The cleaved 6xHis-MBP tag was removed by incubating the solution with Ni-NTA magnetic beads (NEB; S1423) for 1 hour at 4 °C. The solution was then placed in a magnetic rack to separate the bead-bound 6xHis-MBP fraction from MISP. The purified full-length MISP was run in an SDS-PAGE gel to confirm successful cleavage.