Overexpress c43 de3 cells

Protein expression and purification of holotoxins was performed as described previously^{31 (link)}. Briefly, the genes for human LT, CT, H1 hybrid toxin, and H2 hybrid toxin were expressed in OverExpress™ C43 (DE3) cells (Sigma-Aldrich). Cells were grown at 37 °C in TB medium containing chloramphenicol until an OD_{600 nm} of 2.0 was reached. Cells were then induced with l-arabinose and harvested after 3 h. Holotoxins were extracted from the bacterial pellet by inducing periplasmic lysis with polymyxin B sulfate (Sigma-Aldrich). Holotoxins were purified by TALON affinity chromatography using a HiTrap TALON crude column (GE Healthcare, Chicago, IL) and size exclusion chromatography with a HiLoad 16/60 Superdex 200 prep grade column (GE Healthcare) equilibrated with phosphate-buffered saline. To reduce B-pentamer contamination due to partially overlapping peaks, only size exclusion fractions prior to the holotoxin peak maximum were pooled, filtered and stored at 4 °C. The hybrid toxins were characterized by SDS-PAGE analysis, tryptic digestion and mass spectrometry.

Expression was essentially performed
as described previously.^{21 (link)} Briefly, OverExpress
C43 (DE3) cells (Sigma) harboring pARCT5 were grown overnight at 30
°C in Terrific Broth (TB) medium containing 25 μg/mL CAM.
Cultures were diluted 1/50 in TB medium, grown until the optical density
at 600 nm (OD₆₀₀) reached 2.0, and induced with 0.2% l-arabinose (Sigma) at 37
°C. After
3 h, the cells were harvested by centrifugation (6000g, 20 min, 4 °C) and resuspended in 1/40th volume of Talon A buffer (50
mM sodium phosphate pH 8.0, 300 mM
NaCl), supplemented with cOmplete^TM protease
inhibitor cocktail (Roche), 1 mg/mL polymyxin B sulfate (Sigma), and
benzonase (Sigma). This solution was incubated at 37 °C for 15
min with shaking followed by centrifugation (8000g, 20 min, 4 °C). The supernatant (containing the periplasmic
extract) was filtered through a 0.22 μm filter (polyethersulfone
(PES) membrane, VWR) and used immediately for further purification.

The gene for CTB (Uniprot: Q57193) was heterologously expressed in E. coli BL21 (DE3) using a CTB-pET21b +construct. For protein production, cells were grown at 37 °C in LB medium containing ampicillin until OD_{600 nm} of 0.5 was reached. The temperature was reduced to 25 °C and IPTG was added to a final concentration of 0.5 mM to start CTB production.
The genes for CT and CT variants (W88K, H18A, H18AH94A) were heterologously expressed in OverExpress™ C43 (DE3) cells (Sigma) using pARCT5 or pARCT5 derivatives. For protein production, cells were grown at 37 °C in TB medium containing chloramphenicol until OD_{600 nm} of 2.0 was reached. l-arabinose was added to a final concentration of 0.2% (w/v) to start holotoxin production.
Cells were harvested after 14–18 h (CTB) or 3 h (holotoxin) by centrifugation (6900 × g, 20 min, 4 °C) and the pellet was re-suspended in 1/60^th volume of TALON A buffer (50 mM sodium phosphate, 300 mM NaCl, pH 8) with 1 mg of polymyxin B (Sigma Aldrich) per mL, cOmplete™ Protease Inhibitor (Roche) and benzonase (EMD Millipore) and shaken at 37 °C for 15 min. Insoluble debris and cells were removed from the periplasmic extracts by centrifugation (8000 × g, 20 min, 4 °C). The filtered supernatant was directly applied to the TALON affinity column.