FIGURE 1.
Isolation and characterization of purified LOS·MD-2 complexes. A, [13/14C]LOS·His6MD-2 was isolated from Ni FF Sepharose by an imidazole gradient following the reaction of LOS·albumin (1 mg LOS) with conditioned insect medium containing sMD-2 (2 liters). The dialyzed reaction mixture was applied at 4 °C at a flow rate of 3 ml/min. (−−−), absorbance; (___) cpm [14C]LOS. B, radioactive peak containing [13/14C]LOS·His6MD-2 recovered from an imidazole gradient from Ni FF Sepharose was concentrated and further purified by chromatography on Sephacryl S100 (1.6 cm × 100 cm) equilibrated in 20 mm phosphate, 150 mm NaCl, pH 7.1, (−−−), absorbance; (-○-) cpm [14C]LOS. C, imidazole eluate from Ni FF Sepharose (1 μl) and LOS·His6MD-2 isolated from S100 and concentrated 10-fold (1 μl sample) were analyzed by PhastGel SDS-PAGE (10–15%)/Coomassie Blue stain. D, dose-dependent activation of HEK/TLR4 cells was measured by increasing concentrations of (-●-) [3H]LOS·His6MD-2 (25,000 cpm/pmol), and purified [13/14C]LOS·His6MD-2 wt (-▴-) and F126A (-△-). Cellular activation was monitored by accumulation of extracellular IL-8 determined by ELISA. IL-8 results shown are representative of at least two independent experiments, each sample performed in triplicate. The chromatographic profiles are representative of a typical purification experiment (n >5); recovery of [14C]LOS was ≥80%.