Generation and production of 3D6Q44E interface mutants

Mutations were introduced in pTT5 (National Research Council, Canada) containing anti-HIV gp41 antibody 3D6 (Grunow et al., 1988; Felgenhauer et al., 1990) heavy or kappa light chain coding sequences respectively using mutagenic primers and the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, 210518) according to the manufacturer's protocol. The Q44E mutation was used previously to influence V_H:V_L pairing (Igawa et al., 2010). 3D6^Q44E and its interface mutants were produced by transient transfection of HEK293-6E cells (National Research Council, Canada) with two separate pTT5 plasmids for light and heavy chain at a molar ratio of 1:1. Cells were cultivated at 37°C in a 5% CO₂ shaking incubator with humidified atmosphere in FreeStyle™ F17 Expression Medium (gibco, A1383501) supplemented with 4 mM l-Glutamine (gibco, 25030-024), 0.1% Pluronic F68 (gibco, 24040-032) and 50 μg/mL G-418 (gibco, 10131-027). Cells were transfected with 1 μg DNA per 1 mL culture using polyethylenimine (PEI, 25 kDa, linear from Polysciences, Inc., 23966). Two days post transfection (dpt), cells received Tryptone N1 (Organotechnie, 19553) at a final concentration of 0.5%. Five dpt the culture was spun down (300 g, 10 min) and the supernatant containing IgGs was filtered through a 0.45 μm nitrocellulose filter (Merck Millipore Ltd, HAWP04700). The supernatant was purified over a HiTrap Protein A HP column (GE Healthcare, 17-0402-01) using the lab-scale chromatography system ÄKTA purifier (GE Healthcare). Antibodies were eluted from the column by addition of 0.1 M glycine (pH 3.5). A 15 μL of 1 M TRIS (pH9) per 1 mL eluate was added to neutralise the pH. After dialysis against PBS, protein samples were analysed by analytical SEC with Superdex 200 10/300, running buffer PBS + 0.2 M NaCl, 50 μg of protein was loaded. Where indicated, IgGs were purified by preparative SEC using a Superdex 200 16/600 column.