Purification of GST-fusion proteins from E. coli

This is a simple protocol for purifying GST fusion proteins expressed using pGEX vectors in E. coli. The final product will be the fusion protein bound to glutathione-sepharose beads (appropriate for GST pull-down assays, etc.). The proteins can also be eluted from the beads using 10 mM glutathione in Tris buffer.

1. Grow overnight culture of DH5alpha cells containing pGEX construct in 3 ml LB + 100 ug/ml ampicillin at 37 C.

2. Dilute 1 ml of each culture to 100 ml LB + ampicillin pre-warmed to 37 C, and incubate (shaking) until OD ~ 0.6 (~ 2-3 h).

3. Add IPTG to 1mM, incubate 4 h.

4. Transfer cultures to centrifuge bottles, centrifuge 10 min, 6000 xg at 4 C.

5. Discard supernatant, store pellet at -80 C if necessary.

Pellets can be stored long-term at -80 C with little effect on yield.

6. Resuspend pellet in 10 ml of ice-cold lysis buffer.

7. Add 1 ml triton buffer to resuspended cells, mix by inversion, keep on ice.

8. Sonicate (power level #3) 3 x 30 sec, placing on ice 1 minute between sonications.

9. Transfer lysate to centrifuge tubes, centrifuge at 10,000 rpm, 10 min, at 4 C.

10. Transfer cleared lysate to 15 ml falcon tubes and add 100 ul glutathione-sepharose beads (200 ul of a 50% slurry), rotate 1 hr at 4 C.

Glutathione-sepharose beads should be rinsed once with lysis buffer before adding to lysate.

11. Wash beads 4x with 1 ml wash buffer (at 4 C).

After each wash, beads can be pelletted by centrifuging for 1 min at 2000 rpm. The total washing time should be at least 20 min.

12. Wash once with 1 ml bead storage buffer, centrifuge and discard supernatant.

13. Add 400 ul bead storage buffer, store at –20 C.

Buffer Recipes

Lysis buffer: (need 10 ml per 100 ml culture )

  • 20 mM HEPES-KOH pH 8.0
  • 500 mM NaCl
  • 0.1 mM EDTA
  • 200 ul 50x complete protease inhibitors per 10 ml (add fresh)

Triton buffer: (need 1 ml per culture)

  • 20 mM HEPES-KOH pH 8.0
  • 500 mM NaCl
  • 0.1 mM EDTA
  • 10% Triton X100

Wash buffer: (need 4 ml per 100 ml culture)

  • 20 mM HEPES-KOH pH 8.0
  • 500 mM NaCl
  • 0.1 mM EDTA
  • 0.1% Triton X-100

Bead storage buffer:

  • 20 mM Tris/HCl, pH 7.4
  • 50% (v/v) glycerol

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