Bl21 gold de3 competent e coli

The pT7-7 α-syn WT plasmid (a
gift from Hilal Lashuel, Addgene, Watertown, NY, United States^{49 (link)}), with codon Y136 mutated from TAT to TAC to
prevent cysteine misincorporation, was transformed into BL21-Gold
(DE3) competent E. coli (Agilent Technologies,
Santa Clara, CA, USA) according to the manufacturer’s instructions.
Expression of α-syn was induced using 1 mM IPTG at 28 °C
overnight, and ampicillin (100 μg/mL) was included as necessary.
The cells were harvested by centrifugation and resuspended in 20 mM
Tris−HCl and 1 mM EDTA, pH 8.0 and protease inhibitors (Roche,
Basel, Switzerland). One protease inhibitor tablet was added per litre
of culture. α-Syn was then purified as previously described.^{50 (link)} Anion exchange chromatography was performed
using a HiPrep Q HP 16/10 column (Cytiva, Little Chalfont, UK) and
a linear gradient from 0 to 1 M NaCl. Finally, monomeric α-syn
was purified by size exclusion chromatography in PBS (137 mM NaCl,
2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄, pH 7.4) using a HiLoad 16/600 Superdex 75
pg (GE Healthcare, UK). The concentration of the protein was determined
by measuring the absorbance at 275 nm, using an extinction coefficient
of 5600 M⁻¹ cm⁻¹. α-Syn
fibrils were obtained by incubating 60 μM α-syn at 37
°C under quiescent conditions for two weeks. NaN₃ (0.02%)
was added to each sample to prevent the growth of bacteria.