Expression and Purification of Human 6xHis


  • Distilled water
  • LB media
  • Carbenicillin
  • IPTG
  • Imidazole
  • Lysis buffer (50mM NaH2PO4 pH 8.0, 300mM NaCl, 10mM Imidazole)
  • Equilibration buffer (50mM NaH2PO4 pH 8.0, 300mM NaCl, 10mM Imidazole)
  • Wash buffer (50mM NaH2PO4 pH 8.0, 300mM NaCl, 20mM Imidazole)
  • Elution buffer (50mM NaH2PO4 pH 8.0, 300mM NaCl, 250mM Imidazole)
  • Lysozyme
  • PMSF
  • TCEP
  • 10X HyClone PBS
  • Dialysis cassettes (7000 MWCO, 3-12 mL)
  • Amicon cut-off filter tubes (100 kDa)
  • Econo-Column® Chromatography Columns (1.5 x 20 cm, glass chromatography column, max. vol. 35 mL, cross-sectional area 1.77 cm2, manufacturer: Biorad)
  • Ni Sepharose® 6Fast Flow medium (manufacturer GE)



Expression of 6xHis-tagged α-syn in Escherichia coli

  1. Clone the full-length C-terminally 6xHis-tagged human α-syn (NM_000345) into the ampicillin-resistant bacterial expression vector pET-21b (Ndel and HindIII cloning sites). To avoid cysteine mis-incorporation at codon 136 in bacterially expressed αSyn, change the codon at 136-TAC to 136-TAT by site-directed mutagenesis.
  2. Transform competent E. coli BL21(DE3)-pLysS according to the manufacturer’s instructions (Invitrogen, cat# 44-0054). Plate 50µL of the final mixture onto an agar plate with 100µg/mL carbenicillin (Chem-Impex, cat# 00049) and incubate overnight at 37°C to select transformed cells.
  3. To prepare a starter culture, inoculate a single colony of the transformed cells to a 250mL flask (Fisher Scientific, cat# 10-090B) containing 50mL of autoclaved Terrific Broth (Fisher Scientific, cat# BP2468500) with 100µg carbenicillin (TB-100C).
  4. Incubate the flask overnight at 37°C with shaking at 200rpm. On the next day, transfer the 50mL culture into a falcon tube and centrifuge at 5,000 x g for 10min at room temperature (RT).
  5. To grow cells for purification, add 50mL of TB-100C to the bacterial pellet under sterile conditions and resuspend thoroughly. Inoculate a 2L (Fisher Scientific, cat# NC0343699) flask containing 500mL TB-100C with 25mL of the resuspended cells.
  6. Incubate the culture at 37°C with 200rpm agitation. Monitor the bacterial growth by measuring the optical density at 600nm (OD600). Induce the culture with 0.1mM IPTG (Sigma Aldrich, cat# I5502-10G) once OD600 reaches 0.6 to 0.7. Use 52.5µL of 1M IPTG to 525mL culture. After the induction starts, incubate the culture for 6h at 25°C with shaking at 150rpm.
  7. Centrifuge the culture for 30min at 3,000 x g and 4°C. Collect the pellet in a 50mL conical tube (Thermo Scientific, cat# 339653). Wash the bacterial pellet with STE washing buffer (10mM Tris-Cl pH 7.5 (Fisher Scientific, cat# BP152-10), 100mM NaCl (Sigma Aldrich, cat# S3014), 1mM EDTA (Promega, cat# V4231)) using 15mL per liter of culture. Centrifuge for 15min at 5,000 x g at 4°C. Discard the supernatant and store the pellet at -80°C until use.

Purification of 6xHis-tagged α-syn

  1. Re-suspend the pellets in 20mL of lysis buffer per liter of culture (50mM NaH2PO4 pH 8.0 (Sigma Aldrich, cat# S5011), 300mM NaCl, 10mM Imidazole (Fisher Scientific, cat# O3196-500), 0.1mM EDTA, 1mM PMSF (Acros Organics, cat# 215740050) prepared in isopropanol, and 0.1mM TCEP (Sigma Aldrich, cat# C4706) added right before use.
  2. Sonicate the lysate with a 0.5-inch probe tip using the Misonix Sonicator for 5min (30s pulse ON, 30s pulse OFF) at 60% intensity on ice.
  3. Centrifuge the lysate at 12,000 x g for 15min at 4°C in 50mL falcon tubes. Collect the supernatant and centrifuge again at 100,000 x g for 30min at 4°C.
  4. Collect the supernatant and filter through 0.45µm filter (Sigma Aldrich, cat# SLHVR33RS). Store the supernatant on ice.
  5. Prepare a 1.5cm x 20cm glass chromatography column (Biorad, cat# 7374152) with 10 ml of Ni Sepharose 6 Fast Flow slurry (Cytivia, cat# 17531802).
  6. Wash the column with 1 column volume (CV) of Elution Buffer (50mM NaH2PO4 pH 7.4, 250mM imidazole, 300mM NaCl, 0.1mM TCEP) using gravity chromatography.
  7. Wash the column with 5CV of Equilibration Buffer (50mM NaH2PO4 pH 7.4, 10mM imidazole, 300mM NaCl, 0.1mM TCEP).
  8. Load the filtered bacterial supernatant onto the column. Wash the column with 5CV of Washing Buffer (50mM NaH2PO4 pH 7.4, 20mM imidazole, 300mM NaCl, 0.1mM TCEP).
  9. Elute the bound protein with Elution buffer (50mM NaH2PO4 pH 7.4, 250mM imidazole, 300mM NaCl, 0.1mM TCEP). Collect 1.5 ml of fractions in 2mL microcentrifuge tubes (Eppendorf, cat# 022431102) kept on ice.
  10. Check the elution fractions with Bradford to detect total protein. Determine fractions containing αSyn by SDS-PAGE analysis and pool them.
  11. Dialyze the pooled αSyn fractions using a dialysis cassette (7000 MWCO, Thermo Scientific, cat# 66710) against 1XPBS (prepared with 10X HyClone PBS (Cytivia, cat# SH30258.02) and deionized water). Perform 2 dialyses at 4°C (1st dialysis, 10mL sample in 4,000mL of 1XPBS for 16 h; 2nd dialysis, 10mL samples in 2,000mL of 1XPBS for 4h).
  12. After the dialysis, if there is noticeable precipitation, centrifuge the sample at 10,000 x g for 10min and 4°C. Filter the resultant supernatant through 0.22µm filter (Sigma Aldrich, cat# SLGPR33RS).
  13. Filter the supernatant through a 100kDa MWCO filter (MilliporeSigma, cat# UFC910024) using a swinging bucket rotor at 3,220 x g and 4°C for 25min.
  14. Filter the sample through 0.22µm and determine protein concentration using the BCA kit (ThermoFisher, cat# 23225).
  15. Finally, make 500µL aliquots in 1.5mL low binding tubes (Eppendorf, cat# 022431081). Snap freeze in liquid nitrogen and store at -80°C until use.