Purification of GST-fusion protein

 

Purification of GST-fusion protein

This protocol uses Pharmacia glutathione sepharose matrix. Other companies also sells GST beads, e.g. Sigma.

 

1) Spin down cells and resuspend in ice-cold PBS (40 ml per 1 L of culture). Lyse cells by French press. Keep everything on ice unless stated otherwise.

2) Spin to pellet cell debris (12,000g for 10 min.). Remove supernatant and dilute with ice-cold PBS to 100ml. Add 2 ml of 50% glutathione sepharose 4B slurry (see below) to supernatant. Incubate at room temperature for 30 minutes.

3) Centrifuge suspension to pellet sepharose matrix (500g for 5 min.) and remove supernatant (keep everything for analysis later).

4) Wash pellet with 10 ml of PBS. Spin to pellet and remove wash. Repeat wash 2X.

5) Elute protein with 1ml of GEB. Incubate at room temperature for 10 min. Spin to pellet sepharose matrix. Repeat elution step 2X.

6) Add 10-20 units of thrombin to the eluate. Incubate at room temperature for 2 hours. Run gel to check that digest is complete (also run sample from previous steps to check that the binding and elution of protein is satisfactory).

7) Load sample onto a gel filtration column (previously equilibrated with 20mM phosphate buffer) to separate your protein from the GST moiety and thrombin. Run gel to check. If necessary, repeat gel filtration step (after pooling and concentrating fractions). Alternatively the GST may be remove by glutathione sepharose after dialysis or gel-filtration.

8) Pool fractions and concentrate protein for NMR analysis.

 

PBS (phosphate-buffered saline) 140 mM NaCl (8.2 g/L) 2.7 mM KCl (0.2 g/L) 10 mM Na2HP04 (1.78 g/L) 1.8 mM KH2PO4 (0.24 g/L) (pH 7.3)

GEB (glutathione elution buffer) 10 mM reduced glutathione 50 mM Tris-HCl (pH 8.0) (dissolve reduced glutathione in Tris.HCl and stored frozen)

Preparation of Glutathione Sepharose 4B

1) Resuspend matrix in bottle by gentle shaking.
2) Remove 2-4 ml of sepharose and pellet sepharose matrix (500 g for 5 min.)
3) Decant supernatant carefully and wash with 15 - 30 ml of ice-cold PBS (mix by inversion). Spin 500g for 5 min. and decant supernatant.
4) Add 1.5 - 3 ml of PBS to give a 50 % slurry.

See TIBS article " Purification of GST fusion proteins " for more discussion.