GFP nano trap: expression, purification and preparation of the beads



Expression and purification of the GFP-nano trap fusion protein

The GFP nano trap is cloned into a pQE vector with a C-terminal His tag, which allows fast and efficient purification after expression in E.coli. The protein is expressed well at standard conditions, which have not been optimised here.
  • transform competent BL21DE with 0,1 ug plasmid, spread 10 ul on a LB+Amp plate and inocculate a 10 ml LB+Amp culture with the remaining suspension. Cultivate overnight.
  • Inocculate 1l LB/Amp (prewarmed) with the overnight culture (1:100)
  • grow culture at 37o until OD600 approx. 0.5
  • add IPTG to 1 mM
  • grow for 4h at 37oC
  • collect cells, spin 5 min, 6000 rpm, GSA rotor
  • suspend thoroughly in cold 50 ml lysis puffer (first suspend in a small volume), add a spatula tip of DNAse
  • lyse the cells with the microfluidizer in Ficner's lab
  • spin for 15 min, 15000 rpm
  • spin in the ultacentrifuge for 15 min, 40000 rpm
  • apply the supernatant to a regenerated HiTrap-Ni column
  • wash with 10-20 volumes of washing buffer (40 mM imidazol)
  • elute with elution buffer (250 mM imidazol)
  • pool the fractions with protein
  • exchange the buffer with NAP-10 columns
  • apply the sample on a ion exhange column
  • elute with a salt gradient
  • pool the fractions
  • exchange the buffer to coupling buffer


coupling to sepharose beads

The protein concentration should be higher than 1 mg/ml. The protein is covalently coupled to activated sepharose. Please proceed according to the protocol for preparation of affinity columns for antibody purification.


Buffer:

Lysis buffer

50 mM Tris/HCl pH7
100 mM NaCl
20 mM imidazol

wash buffer

50 mM Tris/HCl pH7
100 mM NaCl
40 mM Imidazol

elution buffer:

50 mM Tris/HCl pH
100 mM NaCl
250 mM imidazol

coupling buffer:

50 mM ?? pH
100 mM NaCl