FIGURE 6.
Time-dependent decay of bioactive sMD-2 monomer at 37 °C (A) and at 4 °C (B and C). A, insect cell conditioned medium containing sMD-2 was diluted 20-fold in PBS with 0.1% albumin or in Express Five™ medium. Bioactive sMD-2 was measured by assay of inhibition of [3H]LOS·MD-2 binding to TLR4ECD (as in Fig. 4) before and after 24 h of preincubation of sMD-2 at 37 °C. The results represent the means ± S.E. of three separate determinations. B, a sample of pooled sMD-2 monomer (10 μl) stored at 4 °C was taken at days 2, 21, and 33 after isolation in Express Five™ medium and incubated with [14C]LOS·sCD14 (2 nm) for 30 min at 37 °C in PBS with 0.1% HSA. The fractions were analyzed for [14C]LOS content by liquid scintillation spectroscopy. The reaction mixture was separated by chromatography on Sephacryl S200 in PBS. The profiles shown are representative of at least two experiments and show reduced activity of isolated sMD-2 monomer (i.e. formation of [14C]LOS·MD-2) during prolonged storage of 4 °C. C, samples (40 μl) of pooled sMD-2 monomer taken 33 days after isolation and storage at 4 °C and of sMD-2-rich conditioned insect cell medium (CM) were treated with SDS-PAGE sample buffer under nonreducing conditions, electrophoresed, transferred to nitrocellulose, and then probed for MD-2 using an anti-(His)4 antibody. The markers are Perfect Protein markers (Novagen).