Purification of native lipoproteins by Triton X-114 phase partitioning.

E. faecalis and B. cereus cultures were grown to late exponential phase (OD₆₀₀ of 1.5) in 15 ml of TSB, washed once with TBSE solution (20 mM Tris-HCl, pH 8.0, 130 mM NaCl, and 5 mM EDTA), and frozen until use. Native bacterial lipoproteins were enriched using the Triton X-114 phase partitioning method with some modifications (12). Briefly, the harvested bacterial cells were resuspended in 800 μl TSBE with 1 mM PMSF and 0.5 mg/ml lysozyme at 37°C for 30 min. Cells were disrupted by bead beating using a MagNA lyser (7000 setting, using an equal volume of 0.1-mm zirconia silica beads in 5 20-s cycles with 2-min chilling period on ice in between cycles). Unbroken cells and beads were removed by low-speed centrifugation (3,000 × g for 5 min at 4°C). The beads were washed with a second aliquot of TBSE, and the supernatants were pooled and then supplemented with Triton X-114 to a final concentration of 2% (vol/vol). After incubation at 4°C for 1 h, the mixture was incubated at 37°C for 10 min to induce phase separation. Samples were centrifuged (10,000 × g for 10 min at room temperature), and the upper aqueous phase was removed and replaced with the same volume of TBSE solution. This procedure was repeated twice more, with subsequent incubations shortened to 10 min. The final Triton X-114 phase was chilled on ice and brought to 500 μl with ice-cold TSBE, and the single-phase solution immediately centrifuged (16,000 × g for 2 min at 4°C) to remove insoluble integral membrane proteins. The lipoprotein-enriched fraction was obtained from the supernatant by precipitation with 3 volumes of acetone and overnight incubation at −20°C. Pellets were washed twice with acetone, air dried, and resuspended in 10 mM Tris-HCl, pH 8.0, or directly in SDS-PAGE sample buffer.