Fig 1: FPLC purification of the CTB-SARS-CoV-2-ACE-RBD mucosal vaccine fusion protein: Electro-elution of a 42–50 kDa protein band following acrylamide gel electrophoresis of transformed E. coli BL-21 cell homogenates may contain, in addition to the vaccine protein, bacterial proteins of a similar molecular weight. To further purify the vaccine protein, the electroeluted proteins were separated by fast protein liquid chromatography (FPLC) on two tandem Agilent SEC-5 columns (5 µm, 300 A, 4.6 × 100 mm) packed with 5 µm silica particles coated with a neutral, hydrophilic layer to aid in protein separation, connected in series and run in PBS buffer, pH 7.4, at 0.4 mL/min on a GE Healthcare FPLC system. The column eluate was monitored by UV spectrophotometry at 280 nm, and protein fractions (0.25 mL) were collected manually. The vaccine fusion protein was identified by ELISA assay in the 5.0–6.0 mL column fractions. In the assay, individual column fraction samples were bound to the wells of an Immulon 1B plate (Thermo Fisher Scientific Inc.). The vaccine protein was identified with an anti-His-Tag Mouse Monoclonal primary antibody (Thermo Fisher Scientific Inc. #37-2900) and a goat anti-mouse (H&L) peroxidase secondary antibody (Thermo Fisher Scientific Inc. #62-6520). Slower-moving molecules and molecular debris were eluted between 7.5 and 8.5 mL.
Supplier Page from Thermo Fisher Scientific for 6x-His Tag Antibody