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].