Expression and purification of recombinant proteins

Recombinant ETEC fimbriae-based MEFA and fimbriae subunit proteins were expressed and purified as previously described [21]. Briefly, a single recombinant E. coli colony was subcultured overnight in 2×YT medium (2× Yeast Extract Tryptone) containing kanamycin at a final concentration of 30 μg/mL. Bacteria were then allowed to grow at 37 °C with vigorous shaking at 220 rpm. Then, 2 mL of this overnight subculture were transferred to 200 mL of fresh 2×YT medium supplemented with 30 μg/mL kanamycin. When the optical density of the bacterial culture reached 0.6 at 600 nm (OD₆₀₀), bacterial culture was induced by 1 mM isopropyl-β-d-1-thiogalactoside (IPTG) for an additional 4 h. Bacteria pellets were then harvested by centrifugation at 12 000 rpm for 15 min. Total recombinant protein was extracted with a bacterial protein extraction reagent (B-PER) (Thermo Scientific, Rochester, NY). Extracted recombinant 6× His tagged inclusion body proteins were purified from the total protein extract using protino^○RNi-TED 2000 packed columns (MACHEREY-NAGEL, Germany) according to the manufacturer’s instructions. Purified MEFA protein was examined via 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie blue staining and subsequent western blot with anti-FaeG monoclonal antibody (1:1000), anti-FedF serum (1:4000), anti-FanC serum (1:4000), anti-FasA serum (1:4000), and anti-Fim41a serum (1:4000).