The SHP099 resulting suspension was centrifuged at 12,000 x g and the GAGs present in the supernatant were precipitated with ethanol (85%), dried and resuspended in 1 ml distilled water. The GAG concentration was determined spectrophotometrically as described previously [69]. The partial digestion of HS and CS was performed as described above. Extraction of L. salivarius Lv72 surface proteins and heparin-affinity chromatography

L. salivarius Lv72 was grown until mid-exponential phase, washed twice with buffer A (50 mM Tris–HCl, 150 mM NaCl; pH 7.5) and the bacterial PD0325901 ic50 cell pellet was resuspended in the same buffer containing a commercial cocktail of EDTA-free protease inhibitors (Roche, Basel, Switzerland), 1 mM MgCl2, 5 mg/ml lysozyme (Sigma-Aldrich) and 0.05 U/ml mutanolysin (Sigma-Aldrich) and incubated overnight at 4°C. Cells were mechanically disrupted by repeated passage through a French press (SLM Aminco Inc), the pellet was washed twice with buffer A and subjected to overnight digestion with 5 mg/ml lysozyme in the presence of protease inhibitors at 4°C, followed by incubation with 5% Triton X-100 (Sigma-Aldrich) for 1 h at room temperature. The final solution was centrifugated at 10,000 rpm for

30 min and the supernatant was applied to a 1 ml heparin affinity column (GE, Buckinghamshire, England) connected learn more to a FPLC system (GE). Bound proteins were eluted with a continuous 0 – 2 M NaCl gradient in 50 mM Tris–HCl buffer (pH 7.5) and aliquots of the protein Mannose-binding protein-associated serine protease fractions were used in HeLa/Lactobacillus adherence assays. Those that interfered most were subjected to anion exchange chromatography in a Q-sepharose FF column (GE), eluted with a continuous 0 – 0.5 M NaCl gradient in 50 mM Tris–HCl buffer (pH 7.5) and the resulting fractions were subjected to adherence interference assays as described above. The protein concentrations were determined with the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, USA) following the instructions of the manufacturer. SDS-PAGE [66] was performed in a “Miniprotean III” system (Bio-Rad, Hercules, USA). The proteins were stained with Comassie R-250 blue [70] or with a protein silver staining kit (GE).

The band of interest was excised from the gels, digested with porcine trypsin and the resulting peptides were analyzed by MALDI-TOF/(MS) at the Proteomic Service of the Centro Nacional de Biotecnología (CNB-CSIC, Madrid). Construction of expression plasmids and purification of the oligopeptide permease A protein (OppA) The oppA sequence of L. salivarius Lv72 [BankIt1609288 Lactobacillus KC703973] was amplified using primer pairs deduced from the oppA sequence of L. salivarius UCC118 (LSL_1882). The sequence encoding the OppA signal peptide was omitted to ease protein purification. The PCR product was purified and cloned into the vector pRSET-B digested with NdeI and BamHI (Fermentas, Thermo Scientific). The resulting plasmid was transformed to E.