Purification of TeNT
Recombinant TeNT was purified by NiSO4 IMAC followed by size-exclusion chromatography (SEC) on an AKTApure 25 FPLC system (GEHealthcare). Briefly, post-expression, cells were pelleted at 4500 × g for 15 m. Cell pellets were either stored at -80°C or immediately sonicated on ice in IMAC buffer (50 mM Tris, 300 mM NaCl, pH 8). Post-sonication cell lysate was cleared by centrifugation at 4,500 × g for 15–30 m at 4°C and the supernatant filtered through a 0.45 μm membrane filter (Whatman). The sample, supplemented with 20 mM imidazole, was then applied to a 1 mL high purity Histrap™ NiSO4 column (GEHealthcare) at 1 ml/min. The sample was washed and eluted with IMAC buffer supplemented with 20 mM and 300 mM imidazole, respectively. The eluted sample was concentrated, and buffer exchanged to phosphate buffered saline (PBS) using a Vivaspin20 column (50 kDa MWCO; Sartorious) as described by the manufacturer. Five hundred microlitres of sample was then applied to a Superdex™ 200 increase 10/300 size exclusion column (GEHealthcare) and eluted in PBS.