Lysozyme

The empty pGEX-2TK or pGEX-4T-2 vectors or those encoding a GST-testin variant were transformed in the E. coli BL21(DE3) pLYSstar strain. Bacteria were pre-cultured at 37°C for 4 h in Luria Bertani (LB) medium supplemented with 1% glucose. This pre-culture was diluted (1/25) in LB medium and grown at 37°C until an optical density of 0.6. Recombinant protein expression was induced by isopropyl-β-D-thiogalactopyranoside (1 mM) in LB medium that was supplemented with 10 μM zinc-acetate and grown at 16°C overnight. Bacterial cells were lysed in 25 mM Hepes (pH 7.6), 0.1 mM EDTA, 5 mM dithiothreitol, complete protease inhibitor cocktail (Roche) and 10.000 units/ml lysozyme (Amersham) and sonicated for 5 min on ice. GST or the GST-fusion protein was purified from the clarified lysate (from a 25 ml cell culture) by incubation for 2 h at 4°C with 25 μl glutathione sepharose^™ 4B (GE Healthcare) in buffer A (25 mM Hepes (pH 7.6), 150 mM NaCl, complete protease inhibitor cocktail). After extensive washing in buffer A, the resin was used in the binding assay. See also scheme Fig 2A.

1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was from Avanti Polar Lipids Inc. (Alabaster, AL). Lipopolysaccharides (LPS, rough strains) from Escherichia coli EH100 (Ra mutant), Whatman Nuclepore membrane filters (track-etched polycarbonate membranes, pore diameter 100 nm), octyl glucopyranoside (OG), sarkosyl, poly-l-lysine (PLL) (150 000–300 000 g mol⁻¹), lysozyme, sodium azide, calcium, Trizma base and 1 M magnesium chloride solutions were from Sigma-Aldrich. DNAse I was from Thermo Fisher Scientific. Argon 5.0 and nitrogen 5.0 gases were from Linde Gáz Magyarország Zrt (Budapest, Hungary). Water was purified with a Milli-Q Integral 3 Water Production Unit (Merck Millipore, Billerica, MA). Round mica sheets were from Ted Pella, Inc. (Redding, CA). Detergent adsorbent SM-2 Bio Beads were from Bio-Rad.