Efficient Fab Variant Expression and Purification

Individual mutants for the validation experiment were constructed using the QuikChange XL Mutagenesis kit (Stratagene, San Diego, CA) according to the manufacturer's instructions. Primers for Quikchange were designed such that they matched 18 bp flanking each side of the mutated region. The nucleotide sequence of the mutated region on the primers was designed to minimize nucleotide mismatch with the WT C05 Fab. For the expression of C05 Fab variants in mammalian cells, both light and heavy chains were cloned into the pFuse vector, which was constructed by removing the Fc-encoding sequence from the pFuse-Fc vector (Invivogen, San Diego, CA). The light chain and heavy chain were transfected into 293T cells in a 2:1 molar ratio. A His₆-tag was inserted at the C-terminus of the heavy chain. The supernantants containing the C05 Fab variants were collected 3 days after transfection. Expression of C05 Fab variants in insect cells was performed as described previously for WT C05 Fab21 (link). Purification of C05 Fab variants was performed by Ni-NTA Superflow (Qiagen, Valencia, CA), and subsequently by size exclusion chromatography on a Hiload 16/90 Superdex 200 column (GE Healthcare, Pittsburgh, PA) in 20 mM Tris pH 8.0, 150 mM NaCl, and 0.02% NaN₃.