Expression and Purification of Human α-Synuclein

(Adapted from Volpicelli-Daley et al; doi:10.1038/nprot.2014.143)

Expression in Escherichia coli

  1. Clone α-syn (human full-length α-syn (NM_000345) into the ampicillin-resistant bacterial expression vector pRK172. Expression of α-syn from this plasmid does not require induction by IPTG
  2. Transform plasmids into BL21(DE3) RIL-competent coli (Life Technologies, cat. no. 230245). Transfer a single colony to 4 ml of Terrific medium, and incubate it for 2 h at 37 °C with shaking (200 rpm) to create a starter culture.
  3. Add 0.5 ml of starter culture to each of five 2-liter flasks with 500 ml of Terrific Broth (12 grams per liter of Bacto-tryptone, 24 grams per liter of yeast extract 4% (vol/vol) glycerol, 17 mM KH2PO4 and 72 mM K2HPO4) with carbenicillin (1:1000 of 100g/L stock). Incubate the flasks overnight at 37 °C with shaking (200 rpm).
  4. Spin the bacteria for 30 min, 3000g, 4C. Follow same as his-tag, wash with STE and freeze.
  5. Resuspend the pellet in high-salt buffer (750 mM NaCl, 10 mM Tris (pH 7.6) and 1 mM EDTA) with protease inhibitors (EDTA-free) and 1 mM PMSF (50 ml for 1 liter of culture).
  6. Sonicate with at least a 0.25-inch probe tip at 60% power, for a total time of 5 min (10s pulse on,10s pulse off, 30 pulses). Sonicate 4 times, longer if necessary.
  7. Boil for 15 min to precipitate unwanted proteins (water bath, 95°C, 20 minutes). Cool on ice.
  8. Spin at 15000g for 20 min at 4 °C. Take supernatant and centrifuge that again. (Pellet not needed).
  9. Dialyze the supernatant with 10 mM Tris (pH 7.6), 25 mM NaCl and 1 mM EDTA. (4L of dialysis buffer per 1L of culture; 1:100 ratio).

Purification

  1. Apply protein to a Hi-Trap Q HP anion-exchange column (GE Healthcare Life Sciences) and run a linear gradient ranging from 25 mM NaCl (buffer A) to 1 M NaCl (buffer B). α-syn is eluted at ~300 mM NaCl. Again, collect fractions (~50 fractions at 2 ml each). Combine 10 μl of each fraction with 10 μl of 2× Laemmli buffer and analyze the fractions by PAGE with 4–20% gradient gels, followed by Coomassie staining/destaining.
  2. Dialyze with 10 mM Tris (pH 7.6) and 50 mM NaCl overnight (same 1:100 ratio as before).
  3. Estimate the protein concentration.
  4. Concentrate the fractions to ~30 mg/ml, divide them into aliquots and store them at −80 °C. The yield should be ~30 mg per 1 liter of culture.