The expression and purification of human wild-type (WT) α-syn was performed as previously described27. Briefly, the Escherichia coli strain BL21 (DE3) (Stratagene, La Jolla, CA, USA) was transformed with expression vector pET3a encoding WT α-syn and the bacteria were grown in LB medium to an optical density of 0.8. α-Syn expression was induced by 0.5 mM isopropyl β-d-1-thiogalactopyranoside for 3 h. The cells were lysed by sonication and the cell lysates were clarified by centrifugation at 14,000 × g for 30 min. α-Syn was precipitated by 50% ammonium sulfate at 4 °C. The solution was centrifuged at 4000 × g for 30 min at 4 °C and the resulting pellet was resuspended in 10 mM Tris pH 7.5. The solution was loaded onto a diethylaminoethyl cellulose column, which was eluted by a gradient of 0–500 mM NaCl, and the fractions containing α-syn (eluted at 200 mM NaCl) were heated to 75 °C for 20 min. Then, the solution was clarified by centrifugation at 14,000 × g, loaded onto a Superdex 75 HiLoad 26/60 column (GE Healthcare), equilibrated, and eluted in 50 mM Tris-HCl (pH 7.5) and 150 mM KCl. Pure α-syn (0.2–0.5 mM) in 50 mM Tris-HCl (pH 7.5) and 150 mM KCl was filtered through sterile 0.22 μm filters and stored at −80 °C. The α-syn concentration was determined spectrophotometrically using an extinction coefficient of 5960 M−1 cm−1 at 280 nm. For the fibril formation, α-syn was incubated at 37 °C under continuous shaking in an Eppendorf Thermomixer set at 600 r.p.m. The assembly was continuously monitored in a Cary Eclipse spectrofluorometer (Varian, Inc., Palo Alto, CA, USA) in the presence of Thioflavin T at an excitation wavelength of 440, an emission wavelength of 480, and an average time of 1 s.

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