Shake flask cultures were all performed in MSS medium containing heptakis(2,6-O-dimethyl)β-cyclodextrin [23, 24]. At 36 h, the production of PT was about doubled in strain Bp-WWD (3.77
± 0.53 μg/mL), compared with Bp-WWC (2.61 ± 0.16 μg/mL) and wild-type Alpelisib chemical structure Tohama (2.2 μg/mL) (Table 1), demonstrating that the level of PT expression was a function of the number of copies of the structural gene cluster. FHA in all three recombinant strains was about the same (Table 1). The production of PRN in shake flask cultures of Bp-WWC, Bp-WWD and Bp-WWE in MSS medium was analyzed by densitometry analysis of Western blot results. PRN amount in the clarified culture supernatants and extract of the separated cells at 60°C was assayed. The amount of PRN in cell extract of Bp-WWC and Bp-WWD was similar (2.48 ± 0.10 and 2.31 ± 0.17 μg/mL, respectively). A two-fold increase was found in Bp-WWE (4.18 ± 1.02 μg/mL), again showing a good correlation of the level of prn expression to the gene copy number. In all three
recombinant strains, the fraction of PRN found in the supernatant fraction in these flask cultures was small or negligible (less than 0.1 μg/mL, data not shown). Table 1 PT, FHA and PRN production by strains Bp-WWC and Bp-WWD and Bp-WWE Strain PT (μg/mL) FHA (μg/mL) PRN (μg/mL)** Tohama wt 2.2 ND* ND* Bp-WWC 2.61 ± 0.16 17.75 ± 3.30 2.48 ± 0.10 Bp-WWD 3.77 ± 0.53 14.33 ± 0.50 2.31 ± 0.17 Bp-WWE 4.49 ± 0.83 17.08 ± 2.21 4.18 ± 1.02 *ND = Not determined **The amount in cell extract The values were the mean of 3 independent Glutathione peroxidase experiments with standard Selleckchem EVP4593 deviation except the data for PT of Tohama WT was obtained from two independent experiments Assessment of PT inactivation PT was purified from culture supernatants using a modification of the process published by Ozcengiz  where the initial ammonium sulphate precipitation was replaced by ligand exchange chromatography [26, 27]. The toxicity of the PT toxin from wild type B. pertussis and Bp-WWC (genetically inactivated PT) was analysed and compared by the Chinese hamster ovary (CHO) cell clustering assay
. This assay has a much higher sensitivity than other functional assays reported for PT. The native toxin purified from strain B. pertussis Tohama demonstrated a clustering endpoint at 2.6 pg per well. The genetically-inactivated PT did not click here promote clustering at the highest concentrations of 0.8-1.6 μg per sample obtained in this test (Figure 6). This assay can, therefore, detect toxicity reduction by a factor of 5 × 105 to 1 × 106, despite limitations imposed by the low solubility of PT. This result demonstrated that PT toxin purified from Bp-WWC was successfully inactivated by insertion of five nucleotide replacements resulting in two amino acid replacements in the PT subunit S1. Figure 6 CHO-cell clustering test.