Purification of scFv16 from Insect Cells

Single chain construct of Fab16 (scFv16) was expressed and purified as previously described^{19 (link)}. In brief, a C-terminal 6× histidine tagged scFv16 was expressed in secreted form from Trichuplusia ni Hi5 insect cells using the baculovirus method (Expression Systems), and purified by Ni-NTA (Qiagen) chromatography. Supernatant from baculovirus-infected cells was pH balanced by addition of Tris pH 8.0. The C-terminal 6x His-tag of Ni-NTA eluent was cleaved by 3C protease, and the protein was dialyzed into a buffer consisting of 20 mM HEPES pH 7.5 and 100 mM NaCl. The sample was reloaded onto the Ni-NTA column to capture the cleaved His₆. The flow-through containing scFv16 was collected, concentrated and purified through gel-filtration chromatography in a final buffer (100 mM NaCl and 20 mM HEPES pH 7.5). Monomeric fractions were pooled, concentrated to ~100 mg/ml.