Constructs were over-expressed in BL21(DE3) pLysS strains according to previously published protocols [12 (link), 13 (link)]. Briefly, cells were grown to mid-log phase (0.6–0.8 O.D.600 nm) and then induced with 0.5 mM IPTG. After 3 h of induction, cells were harvested by centrifugation at 8000 rpm for 20 min at 4° in a JLA 8.1 rotor.
Cell pellets were lysed using C3 Emulsiflex homogenizer (Avestin Inc., Otowa, ON, Canada) at 18 000–20 000 psi. The lysate was clarified using a Beckmann Optima X-100 Ultra centrifuge at 100 000 x g for 60 min The clarified lysate was run over a HisTrap HP NiNTA column (Cytiva) on an AKTA Pure system (Cytiva). Prior to removal of Hisx6 tags by TEV protease, the CPP fusions were further purified by size exclusion chromatography (SEC) on a HiLoad 16/600 Superdex 200 pg (Cytiva). Cleaved proteins were quantified using UV/visible spectroscopy on a Perkin Elmer Lambda 360 using the extinction coefficients calculated for each sequence by ExPasy ProtParam [37 (link)].
TEV protease [38 (link)] was added to purified constructs in a 1:10 ratio and incubated at 4 °C with light rocking overnight. After NiNTA resin (BioRad) was added, the solution rocked at room temperature for 15 min, followed by centrifugation at 1000 x g, and the supernatant containing cleaved constructs was collected.
Cell pellets were lysed using C3 Emulsiflex homogenizer (Avestin Inc., Otowa, ON, Canada) at 18 000–20 000 psi. The lysate was clarified using a Beckmann Optima X-100 Ultra centrifuge at 100 000 x g for 60 min The clarified lysate was run over a HisTrap HP NiNTA column (Cytiva) on an AKTA Pure system (Cytiva). Prior to removal of Hisx6 tags by TEV protease, the CPP fusions were further purified by size exclusion chromatography (SEC) on a HiLoad 16/600 Superdex 200 pg (Cytiva). Cleaved proteins were quantified using UV/visible spectroscopy on a Perkin Elmer Lambda 360 using the extinction coefficients calculated for each sequence by ExPasy ProtParam [37 (link)].
TEV protease [38 (link)] was added to purified constructs in a 1:10 ratio and incubated at 4 °C with light rocking overnight. After NiNTA resin (BioRad) was added, the solution rocked at room temperature for 15 min, followed by centrifugation at 1000 x g, and the supernatant containing cleaved constructs was collected.
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