Expression, purification and labeling of α-synuclein

Recombinant full-length and truncated (1–108) α-synuclein with and without ¹⁵N enrichment were expressed in E. coli (BL21-DE3) and purified as described⁴⁷. Five microliters of pT7-7 plasmids containing the human α-synuclein insert were transformed into 100 μl of BL21-DE3 cells. Positive transformants were grown overnight, inoculated into 1 L of LB or M9 media supplemented with ¹⁵NH₄Cl, and grown until the optical density reached 0.6 at 600 nm. Expression of α-synuclein was induced with 0.5 mM (final concentration) of IPTG. After 4 hours, cells were harvested by centrifugation at 5000 rpm for 15 min. The pellet was resuspended in 10 mM Tris-HCl, pH 8, 1 mM EDTA, 1 mM PMSF and frozen with liquid nitrogen. Cells were lysed by thawing in a 70 °C water bath and sonication. After centrifugation to remove cell debris, DNA was precipitated by streptomycin sulfate (10 mg/mL), and α-synuclein, by ammonium sulfate (361 mg/mL). The protein pellet was resuspended in 25 mM Tris-HCl, pH 7.5, and α-synuclein was purified by ion-exchange chromatography on the Akta Purifier System (GE Healthcare) equipped with a POROS HQ column (Applied Biosystems) and Unicorn 5.11 software. Truncated (1–108) α-synuclein was recovered in the flow-through of anion-exchange chromatography, loaded onto a Superdex 200 10/300 (GE Healthcare Life Sciences) column equilibrated with 25 mM Tris-HCl, pH 7.5, and eluted at 0.5 mL/min. Proteins were quantified by measuring their absorbance using the coefficient of extinction at 275 nm of 5600 M⁻¹ cm⁻¹ (full-length protein) and 1490 M⁻¹ cm⁻¹ (truncated version). A protein aliquot was analyzed by SDS-PAGE on 4–12% Bis-Tris gel (Novex, Life Technologies cat#NP0335) and probed with monoclonal antibodies against human α-synuclein (BD Biosciences, cat#610786 and Cell Signaling, cat#2628) (Supplementary Fig. ^S1J). For improved resolution, the purified protein was electrophoresed on a 16% Tricine gel (Invitrogen cat# EC6695) and stained with Coomassie blue. Purity of the protein was assessed by SDS-PAGE and judged to be greater than 90%.