Captureselect kappaxl

Unique Fab hits from FBC panning, as assessed by VH and VL sequencing, were sub-cloned into a modified pET11a expression vector by asymmetric Sfil cloning.^{56 (link)} This vector introduces a His₆ tag onto the C-terminus of the Fab heavy chain. These plasmids were transformed into E. coli strain Rosetta(DE3) (EMD Millipore) using a classical calcium chloride method. Fabs expression was conducted by growing transformed bacteria in autoinduction cultures containing 100 µg/mL carbenicillin and 25 µg/mL chloramphenicol overnight at 30°C with shaking at 250 rpm. Periplasmic extracts from these Fab expression cultures were generated^{55 (link)} and then IMAC- purified using a 1-mL HisTrap HP column (GE Healthcare) as previously described.⁵⁷ A portion of the I MAC purified Fabs were biotinylated via Sortase A ligation using the following reaction conditions: 20 µM Fab (1 mg/ml_), 2 μΜ Sortase A, 250 μΜ Gly₃-biotin, and 10 mM CaCI₂ in TBS. After incubating for 3 h at 42°C, biotinylated Fabs were purified using CaptureSelect Kappa-XL or CaptureSelect LC-lambda (Hu) affinity matrices (Thermo Fisher Scientific) per the manufacturer’s protocol. Fabs were then concentrated and buffer exchanged to TBS using Amicon Ultra-0.5 mL 10 kDa Centrifugal Filter Units (EMD Millipore). The quality of Fabs was analyzed by SDS-PAGE. Absorbance at 280 nm was used to quantify purified Fab concentration.