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S100A9 is a Damage Associated Molecular Pattern (DAMP) that activates the innate immune system via Toll-like receptor 4 (TLR4). Despite many years of study, the mechanism of activation remains unknown. To date, much of the biochemical characterization of S100A9 has been performed using recombinant S100A9 expressed in E. coli (S100A9 ec ). TLR4 is the canonical receptor for LPS, a molecule found in the outer membrane of E. coli , raising the possibility of artifacts due to LPS contamination. Here we report characterization of LPS-free recombinant S100A9 expressed in insect cells (S100A9 in ). We show that S100A9 in does not activate TLR4. This difference does not appear to be due to LPS contamination, protein misfolding, purification artifacts, or differences in phosphorylation. We show instead that S100A9 in adopts an altered oligomeric state compared to S100A9 ec . Disrupting oligomer formation with the E. coli disaggregase SlyD restores activity to S100A9 in . Our results also indicate that the oligomeric state of S100A9 is a major factor in its ability to activate TLR4 and that this can be altered in unexpected ways by the recombinant expression system used to produce the protein.