Aliquots of 40 μg of protein were loaded onto sodium dodecyl sulphate (SDS) polyacrylamide gels using a final polyacrylamide concentration of 10% or 12.5% w/v (Laemmli 1970). Proteins were resolved at a constant voltage of 170 V and visualized by GelCode Blue staining (Pierce). Selected bands were excised from gels and digested with trypsin using standard protocols, the resulting peptide mixture being analysed by tandem MS (MS/MS). Data were acquired using

a MALDI Q-Tof Premier mass spectrometer (Waters, Manchester, UK), with α-cyano-4-hydroxy-cinnamic acid (Sigma-Aldrich) used as a matrix (3.6 mg mL−1 solution in 50% acetonitrile in 0.1% v/v aqueous trifluoroacetic acid TFA). Monoisotopic masses were corrected using the pseudomolecular ion of Glu-fibrinopeptide

as BMN 673 price a lock mass (1570.6774 Da). Proteins were identified using the MS/MS search module from mascot software (http://www.matrixscience.com) Crenolanib datasheet against the nonredundant protein NCBI database, using the monoisotopic masses derived from trypsinolysis. The following parameters were used: peptide charge +1, peptide tolerance ± 0.1 Da, MS/MS tolerance ± 0.1 Da and one missed cleavage allowed for trypsin. Gels were repeated three times from independent cultures. Flagellin was amplified from B. cereus CH total DNA, which was extracted using the DNeasy Blood and Tissue Kit following the manufacturer’s instructions (Qiagen S.A., Courtaboeuf, France), with the primers FBC-Dir: 5′-GGGGCGCCGGCATGGATTTTTTCGCATATTAC-3′ and FBC-Rev: 5′-CGGGGGCCGGCCTATTGTAATAATTTAGAAAC-3′, in which NaeI sites are shown underlined. The relevant sequence was cloned into the blunt-end NaeI site of a pNZ8048-based lactococcal vector denominated pNZ8110, under the control of the nisin A-inducible promoter PnisA (de Ruyter et al., 1996). In addition, pNZ8110

carries just after PnisA an in-frame sequence coding from the signal peptide of the lactococcal protein Usp45, which allowed flagellin secretion (van Asseldonk et al., 1990). The resulting plasmid, ASK1 denominated pNZ8110-CH, was used to transform L. lactis ssp. cremoris NZ9000 by electroporation. This strain carries chromosomal nisRK genes needed for the nisin-induced activation of PnisA. The plasmid insert was sequenced to be sure that no undesirable mutations were introduced (GenBank accession HQ262412). Because of instability of the recombinant flagellin, the plasmid was transferred to L. lactis ssp. cremoris SMBI198, which are derived from strain NZ9000 by a deletion in the chromosomal htrA gene (Poquet et al., 2000; Rigoulay et al., 2004). The resulting strains, L. lactis ssp. cremoris CH, produced exclusively a surface-associated recombinant flagellin. Flagellin production was induced in L. lactis ssp. cremoris CH cultures at an A600 nm of 0.3 using a concentration of 33 ng mL−1 nisin.