Cecal contents of R. gnavus E1 monoxenic rats were diluted in phosphate-buffered saline (PBS) (10 g for 30 ml) supplemented with a protease inhibitor cocktail (cOmplete ULTRA tablets EDTA-free, Roche). The suspension was centrifuged at 10,000g for 15 min. Resuspension and centrifugation were repeated twice in half the initial volume of PBS. The three supernatants were pooled and filtered on a polyethersulfone membrane with a 10-kDa cutoff (Merck). The resulting filtrate, containing small molecules including peptides, was submitted to ion exchange chromatography using a Carboxymethyl Sepharose column (GE Healthcare) at 4°C. Elution was performed at 2 ml/min with 20 mM acetate sodium buffer at pH 6.5 under isocratic conditions for 36 min, followed by a linear 0 to 0.5 M sodium chloride gradient for 60 min with a detection at 214 and 280 nm. Elution fractions were desalted by DSC-18-SPE columns (Sigma-Aldrich). Briefly, samples were loaded after dilution (1:1) in 0.1% trifluoroacetic acid (TFA), washed with 0.1% TFA, and eluted in 90% acetonitrile (ACN) and 0.1% TFA before being lyophilized and resuspended in water. Fractions displaying anti-Cp activity (as described below) were further purified by size exclusion chromatography using a Superdex 30 Increase column (GE Healthcare). Elution was performed at 4°C at 0.5 ml/min for 120 min under isocratic conditions with PBS, and collected fractions were desalted as described above. Active fractions were then applied to a Jupiter 15-μm C18 300 Å analytical reverse-phase HPLC column (250 mm by 21.2 mm; Phenomenex). Peptides were eluted at 1 ml/min with a 0 to 40% linear gradient of 90% ACN and 0.1% TFA for 30 min before being lyophilized and resuspended in water. The different fractions were maintained at 4°C during all purification steps and stored at −20°C. The protein concentration was estimated either by the Bradford coloration method using a bovine serum albumin standard curve or by measuring the A280 and using the theoretical extinction coefficient of 8480 M−1 cm−1.

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