Superdex 200 pg

The genes encoding the selected scFv clones were cloned into a modified mammalian expression vector containing the hIgG₁ Fc regions (hFc) at the C-terminus as described previously [67 (link)]. The expression vectors were transfected into HEK293F cells (Invitrogen), and the fusion proteins were purified by Protein A affinity chromatography as described previously [67 (link)].
For the expression of IgG₁, genes encoding V_H and V_L were amplified from the phage clones, cloned into a mammalian expression vector, and transfected into HEK293F cells. Then, IgG₁ was purified by Protein A affinity chromatography as described previously [68 (link)]. Then the eluate containing IgG₁ was subjected to gel filtration chromatography. A total of 4 mg of IgG₁ was injected at a flow rate of 1 mL/min and purified by gel filtration using a XK16/100 column packed with Superdex 200 pg at pH 7.4 (ÄKTA pure, GE Healthcare). The chromatogram was recorded at a UV absorbance of 280 nm. The fractions containing IgG₁ were pooled by collection criteria and concentrated.

The cells expressing the protein of interest were harvested by centrifugation at 4000 rpm for 10 min at 4°C. The cell pellet was suspended in immobilized metal affinity chromatography (IMAC) binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris) pH 7.5. The lysate was prepared using a microfluidizer at 15,000 psi for three cycles and clarified by centrifugation at 10,000 rpm for 30 min at 4°C. The clarified supernatant was applied to a Ni²⁺-charged IMAC column with chromatography carried out on an AKTA™ purification system (GE Healthcare, Chicago, IL). The loaded column was washed with additional IMAC binding buffer and then elution carried out using an elution buffer (0.5 M imidazole, 0.5 M NaCl, and 20mM Tris, pH 7.5). The His₆ tag was removed using TEV protease after overnight dialysis in IMAC binding buffer. The protein was then applied to a second Ni-IMAC column and the flow-through collected, concentrated and buffer exchanged into 20 mM Tris, 200 mM NaCl, pH 7.5 prior to loading onto an equilibrated Superdex 200 pg (GE) for size exclusion chromatography (SEC). All fractions were analyzed by SDS-PAGE. SEC was also used to purify Spa33-MxiN complexes after mixing the proteins after each had been purified separately. TheMxiN, Spa33-MxiK and Spa33-MxiN samples were made to 10% (v/v) glycerol.