Removing the noise: endotoxin-free α-synuclein for accurate in vitro screening of fibrillation modulators.

The presence of lipopolysaccharide (LPS) contaminant from the vector expression bacteria Escherichia coli was described as a factor responsible for accelerating the in vitro fibrillation of recombinant α-synuclein (αS), a key protein involved in the neuropathogenesis of Parkinson's disease. Herein, we describe an efficient protocol for the removal of LPS from αS samples based on phase separation using Triton X-114. Importantly, this method was more feasible, faster and cheaper in eliminating LPS than the commercially available protocol using an affinity matrix of modified polymyxin B. Endotoxin-free αS (αSLPS-free) and the protein purified by a commonly used method (αSc) were evaluated in terms of the kinetics of fibrillation, the microstructure of the aggregates and their susceptibility to certain inhibitors of fibrillation. Monomeric αSc and αSLPS-free exhibit quite different susceptibility to modulators of the fibrillation process, despite their fibrils having similar morphologies and thickness according to the atomic force microscopy analysis. Unlike αSc, the fibrillation of αSLPS-free was poorly inhibited to low concentrations of rosmarinic acid or baicalein. On the other hand, αSLPS-free, but not αSc, had its fibrillation accelerated by harman and rifampicin. Collectively, these results highlight that the presence of LPS might generate artefacts regarding the in vitro screening of fibrillation modulators.