Expression of uniform [13C, 15N] labeled wild-type Asyn was carried out in E. coli BL21 (DE3)/pET28a-AS in modified Studier medium M (Studier 2005 (link)). The labeling medium contained 3.3 g/L [13C]glucose, 3 g/L [15N]ammonium chloride, 11 mL/L [13C, 15N]Bioexpress (Cambridge Isotope Laboratories, Inc., Tewksbury, MA), 1 mL/L BME vitamins (Sigma), and 90 μg/mL kanamycin. After a preliminary growth in medium containing natural abundance (NA) isotopes, the cells were transferred to the labeling medium at 37 °C to an OD600 of 1.2, at which point the temperature was reduced to 25 °C and protein expression induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and grown for 15 h to a final OD600 of 4.1 and harvested.
Protein purification was done as described previously (Barclay et al. 2018 (link)). Briefly, cells were lysed chemically in the presence of Turbonuclease (Sigma) to digest nucleic acids. Purification began with a heat denaturation of the cleared lysate, followed by ammonium sulfate precipitation (Kloepper et al. 2006 (link)). The resolubilized protein was bound to QFF anion exchange resin (GE Healthcare Life Sciences, Marlborough, MA) and eluted using a linear gradient of 0.2–0.6 M NaCl. Fractions containing Asyn monomer, which eluted at about 0.3 M NaCl, were pooled, concentrated, and run over a 26/60 Sephacryl S-200 HR gel filtration column (GE Healthcare Life Sciences) equilibrated in 50 mM Tris-HCl, 100 mM NaCl, pH 8 buffer. Fractions were pooled, concentrated to ~20 mg/mL Asyn, and dialyzed at 4 °C into 10 mM Tris-HCl pH 7.6, 50 mM NaCl, 1 mM DTT, and stored at a concentration of ~14 mg/mL at −80 °C until use. Yields were 95 mg purified AS protein/L growth medium for the uniform [13C, 15N] labeled monomer.
Protein purification was done as described previously (Barclay et al. 2018 (link)). Briefly, cells were lysed chemically in the presence of Turbonuclease (Sigma) to digest nucleic acids. Purification began with a heat denaturation of the cleared lysate, followed by ammonium sulfate precipitation (Kloepper et al. 2006 (link)). The resolubilized protein was bound to QFF anion exchange resin (GE Healthcare Life Sciences, Marlborough, MA) and eluted using a linear gradient of 0.2–0.6 M NaCl. Fractions containing Asyn monomer, which eluted at about 0.3 M NaCl, were pooled, concentrated, and run over a 26/60 Sephacryl S-200 HR gel filtration column (GE Healthcare Life Sciences) equilibrated in 50 mM Tris-HCl, 100 mM NaCl, pH 8 buffer. Fractions were pooled, concentrated to ~20 mg/mL Asyn, and dialyzed at 4 °C into 10 mM Tris-HCl pH 7.6, 50 mM NaCl, 1 mM DTT, and stored at a concentration of ~14 mg/mL at −80 °C until use. Yields were 95 mg purified AS protein/L growth medium for the uniform [13C, 15N] labeled monomer.
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