Purification of GST fusion proteins


  1. Grow ~200ml overnight culture of bugs containing desired fusion construct. Inoculate 4 x 1.5 LB/Amp (for total 6l0 with ~50 ml of the overnight.

  2. Grow cultures at 370C to OD600 ~ 0.6. Add IPTG to final concentration of 0.25mM and continue to incubate at 370C (or 300C for highly insoluble protein) for 2-4h to induce fusion protein.

  3. Harvest cells by centrifugation, resuspend in a small volume of PBS (keep cells on ice during resuspension) and pellet in CSA bottle. Freeze pellets in liquid nitrogen. Frozen pellets may be stored at -800C.

  4. Resuspend frozen pellet in ~5 volume PBS+lysozyme. EGTA, EDTA and protease inhibitors (see below). Resuspend either by pipetting up and down or by stirring on a magnetic stirrer (in a cold room) or both.

  5. Sonicate, 3 x 45s pulses. Keep cells on ice between and if possible, during pulses of sonification.

  6. Add KCl to final concentration of 0.25M and DTT to 15mM. Spin lysate 1h at 40K in Beckman 70Ti rotor. Collect supernatant.

  7. Load supernatant from 7 onto ~5-10ml glutathione agarose column equilibrated in PBS + 0.25 M KCl + 0.5 mM DTT (this is buffer A if using FPLC) at flow rate of 0.3 ml/min. If time permits, re-load flow-through onto column 9binding is rather inefficient). Wash with the same buffer until A280 is flat, baseline.

  8. Elute bound proteins with buffer B: 50mM Tris-Cl, pH 8.0, 250 mM KCl, 5mM glutathione. Collect 1ml Fractions and check by SDS-PAGE.

  9. Pool peak fractions and dialyze extensively against dialysis buffer C (3-4 changes, 100 volumes each, over 4-6h).

  10. Measure protein concentration. Make aliquots and freeze in liquid nitrogen. Store at -800C


Reagents

Glutathione-Agarose (Sigma G-4510)

0.25M reduced glutathione in water

20mg/ml lysozyme stock

PBS 2l

70mM Na2PO4 19.9g

68.2mMNaH2PO4 18.9g

1.8mM KH2PO4 0.489g

138mMNaCl 16.2g

2.7mMKCl 0.403

PBS + lysozyme, DTT, protease inhibitors

Same as PBS above + the following components, added just prior to use:

1mM EDTA

1mM EGTA

1mM PMSF

1mg/ml leupeptin

2mg/ml aprotinin

200mg/ml lysozyme


PBS + 0.25 M KCl, 0.5 mM DTT (Buffer A)

Same as PBS above+

0.25M KCl;

0.5mM DTT

Elution Buffer (Buffer B) 250ml

50mM Tris-Cl, pH 8.0 12.5ml 1M

O.25M KCl 4.66g

5mM glutathione 384mg

Dialysis Buffer (C) 2l

50mM Hepes, pH 7.6 100ml 1M

50mM KCl 7.46g

30% glycerol 600ml

1mM DTT 2ml 1M