2.2 | Expression and purification of recombinant EspB
EPEC O127:H6 strain E2348/69 deleted for the espB gene
(ΔespB) (Luo & Donnenberg, 2006) was transformed with a
bacterial expression vector encoding His-tagged EspB (EspB-His) and
grown overnight in Luria-Bertani (LB) broth supplemented with the
appropriate antibiotics. The overnight culture was diluted 1:50 and
grown for 3 hr under T3SS-inducing conditions (pre-heated Dulbecco’s
modified Eagle’s medium [DMEM] in a tissue culture incubator with
5% CO2, statically). These conditions induce the
secretion of EspB into the extracellular environment. Next, 0.5 mM
isopropyl-β-d-thiogalactopyranoside (IPTG) was added, and the
culture was grown for an additional 4 hr. The culture was centrifuged
for 30 min at 12000 × g , and the supernatant containing the
secreted EspB-His was collected and supplemented with protease inhibitor
cocktail of 200 mM phenylmethylsulfonyl fluoride (PMSF) and 1 µM
benzamidine. The supernatant was then loaded on a His-Trap HP 1‑mL
column (GE Healthcare), washed with 50 mM imidazole, and eluted with 500
mM imidazole, according to the manufacturer’s protocol. The elution
fractions were analyzed by SDS-PAGE and Coomassie staining to identify
the fractions that contain the purified protein. The recovered protein
was further purified by gel filtration chromatography using a Superose
12 10/300 GL column (GE Healthcare). The peak fractions were collected,
frozen in liquid nitrogen and stored at ‑80°C.