Purification of GST fusion proteins


  • transform BL21DE with the GST plasmid. Do not store the transformed cells for longer than one week. BL21DE are genetically pretty unstable.
  • Induce expression of the GST fusion protein (standard conditions:  BL21DE, 0,1 mM IPTG, induction at OD=0,5, 18°C overnight)
  • prepare a cleared lysate (2-4 ml lysis buffer per g wet weight of E. coli, lysis with french press, spin 20 min, 10k, UZ spin 20 min, 40 k)
  • equililibrate the column with 10 volumes of lysis buffer (flow rate: 1 ml/min)
  • load the cleared lysate (flow rate 0,5-1 ml/min)
  • wash with 10-20 volumes of lysis buffer (until the OD280 does not change anymore)
  • optional: wash with 10 volumes of stringent washing buffer (e.g. with additional 500 mM NaCl)
  • elute with elution buffer with 10 mM GSH (flow rate 0,5 ml/min, fraction size 0,5 ml, about 10 volumes)
  • regenerate the column
  • confirm the UV profile with an amidoblack test of the fractions (2 µl spots on nitrocellulose)
  • pool the fractions with the highest protein concentrations
  • dialyse overnight (3x3h with 100x sample volume)
  • test whether any precipitate formed, spin the sample 10 min 10k
  • determine protein concentration by Bradford assay
  • add glycerol to 50% or sucrose to 200 mM
  • snap freeze  aliquots in liquid nitrogen and store at -70°C

buffer:

lysis buffer:

  • PBS
  •  50 mM Tris/HCl pH8, 100 mM NaCl, 1 mM DTT

wash buffer:

  • PBS, additional  500 mM NaCl
  • 50 mM Tris/HCl pH8, 500 mM NaCl, 1 mM DTT

elution buffer:

50 mM Tris/HCl pH 8, 50 mM NaCl, 10 mM Gluthathion
prepare aliquots of 5 ml 100 mM Gluthathion and freeze at -20°C. Only before use dilute these aliquots into 50 ml elution buffer.
Check pH! Do not store the 1x elution buffer.