Purification and aggregation of recombinant αS
Expression and purification of recombinant human αS was performed as previously described [7,34]. The purity of the recombinant αS protein was evaluated by SDS-PAGE electrophoresis and protein samples were then applied to a Pierce Spin column (#88275; Thermo Scientific) to remove contaminating endotoxins. The Limulus Amebocyte Lysate assay revealed that monomeric αS stock solutions contained less than 0.1 endotoxin unit (EU)/mg protein at this stage. The protein solutions were then filtered and centrifuged, and the protein content of the supernatant was measured at 280 nm with a Nanodrop 8000 spectrophotometer (Thermo Fisher Scientific). The protein solution was finally adjusted to obtain appropriate concentrations.
To produce αSa fibers, recombinant αS samples were incubated at 37oC under orbital agitation for 96 h (Thermomixer comfort; Eppendorf; Montesson, France). Protein aggregates were harvested, sonicated for 2 min and kept at -20oC until further use as previously described [7]. The production of amyloid fibrils through this protocol was previously confirmed by transmission electron microscopy [30,35].