Pfastbac 6xhis mbp lic expression vector

The full length human MISP sequence was originally obtained from Harvard PlasmID Database (HsCD00326629). MISP was then shuttled into Gateway-adapted plasmid pEGFP-C1. To create baculovirus expression vectors, EGFP-MISP sequence was subcloned into a modified pFastBac-6xHis-MBP LIC expression vector (Addgene; plasmid #30116). In-frame sequence insertion was confirmed by sequencing. The 6× His-MBP-EGFP-MISP construct was expressed and purified from Sf9 insect cells as previously described (30 ). Briefly, 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). The resultant lysate was then centrifuged at 35,000 rpm in a Ti 50.2 rotor (Beckman) for 30 min at 4 °C. Samples were loaded into a HisTrap column according to the manufacturer protocol and eluted with a 50 to 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) in storage buffer (20 mM Tris HCl, 0.1 M KCl, 10 mM imidazole, 10% glycerol, 1 mM EGTA, 2 mM DTT, pH 7.5).