FIGURE 4.
Isolation of TLR4ecd, evaluation of reactivity with LOS·MD-2, and preparation of LOS·MD-2·TLR4ecd complexes. A, monomeric His6TLR4ecd was isolated on Sephacryl S200 (1. 6 cm × 100 cm) at 4 °C at a flow rate of 0.5 ml/min in 20 mm phosphate, 150 mm NaCl, 0.005% BSA, pH 7.1 after application of the concentrated imidazole eluate fractions that contained His6TLR4ecd recovered from the Ni FF Sepharose column. Fractions (2 ml) from the Sephacryl S200 were collected and tested for immunoreactivity with an anti-His antibody; a blot of fractions containing His6TLR4ecd is shown. B, the reactivity of the purified TLR4ecd (5 μl of the S200 peak) was evaluated by reaction with [3H]LOS·MD-2 (1.1 nm) in PBS, pH 7.4. Samples were incubated in 0.5 ml for 15 min at 37 °C and reaction products were resolved on S300 Sephacryl (1.6 cm × 70 cm) in PBS, 0.1% HSA, pH 7.4. Radioactivity in eluate fractions was determined by liquid scintillation spectroscopy. The experiment shown is a representative chromatogram; elution peaks of IgG (158,000), BSA (65,000), and LOS·MD-2 (25,000) are indicated. C, molar excess (1.2×) of purified TLR4ecd was incubated with [13/14C]LOS·MD-2 wt or F126A for 30 min at 37 °C; an aliquot of the reaction mixture was analyzed by size exclusion on Sephacryl S300 equilibrated in 20 mm phosphate, 150 mm NaCl, pH 7.1. The amount of radioactivity in each fraction was evaluated by liquid scintillation spectroscopy. The data shown here are representative of multiple similar experiments (n >4).