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MOLECULAR BIOLOGY: WORKING WITH DNA

EXPRESSION: FUSION PROTEINS

Purification of GST-Fusion Proteins from E. coli-Alternate Protocol 2

Purification of GST-Fusion Proteins from E. coli-Alternate Protocol 2
 
Overview
The protein of interest is cloned in frame behind the coding sequence for the Glutathione S Transferase gene to create a GST-fusion protein. This construct is transformed into bacteria, and the heterologously expressed fusion protein is purified through its affinity for Glutathione-Agarose.
 
Procedure
1. Inoculate 20 ml of LB Medium containing Ampicillin with a bacterial colony transformed with a plasmid containing the GST-fusion protein construct of interest.

2. Incubate overnight at 37°C with vigorous shaking.

3. Add the 20 ml culture to 180 ml of LB Medium with Ampicillin.

4. Incubate for 2 hr at 37°C with vigorous shaking.

5. Add 40 μl of 0.5 M IPTG to induce transcription of the plasmid and incubate for four more hours.

6. Cool the bacterial culture on ice for 15 min.

7. Pellet the bacteria by centrifuging at 4,000 X g for 10 min at 4°C.

8. Resuspend the bacterial pellet in 10 ml of Lysis Buffer.

9. Freeze cell suspension at -80°C.

10. Thaw cells in an ice-water bath.

11. Sonicate the lysate four times with 15 sec bursts. Keep the lysate on ice during sonication and leave the lysate on ice for 1 to 2 min between bursts.

12. Centrifuge at 30,000 rpm in a SW40 rotor (113,650 X g) for 15 min at 4°C.

13. During the centrifugation step, pellet 1.33 ml (1 ml bed volume) of Preswollen Glutathione-agarose (Pharmacia) by centrifugation at 500 X g for 5 min.

14. Wash the Agarose beads with 10 ml of PBS.

15. Pellet the glutathione-agarose beads by centrifuging at 500 X g for 5 min. Remove supernatant.

16. Resuspend the beads to 1 ml with PBS.

17. Transfer the bacterial lysate supernatant to a fresh 50 ml conical tube.

18. Add 1 ml of prewashed glutathione beads (from Step #16) to the lysate.

19. Incubate for 1 hr at 4°C with rotation.

20. Pellet the Glutathione-Agarose beads by centrifugation at 500 X g for 5 min.

21. Wash the beads four times with 10 ml of ice-cold PBS. Pellet the beads between washes by centrifugation at 500 X g for 5 min.

22. Elute the bound GST-fusion protein by incubating the Agarose beads in 1 ml of Elution Buffer for 10 min at room temperature.

23. Centrifuge at 500 X g for 5 min to pellet the Agarose beads.

24. Transfer the eluate (supernatant) to a fresh tube.

25. Repeat the elution (Steps #22 - #24) at least five more times. Keep the eluates separate.

26. Determine which eluates contain protein (see Protocol ID#238) or separate each eluate sAmpicillinle on a 10% SDS polyacrylamide gel (see Protocol ID#455).

27. Pool the appropriate eluates.

28. Dialyze overnight against PBS.

29. Store the purified protein at -80°C. Glycerol may be added as a cryoprotectant.

Solutions
Elution Buffer   50 mM Tris-Cl, pH 8.0
5 mM Glutathione
0.5 M Glutathione   Store aliquots at -20°C
Filter sterilize
0.15 g/ml Glutathione
PBS   pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl
0.5 M Isopropyl beta-D-thiogalactopyranoside (IPTG)   0.12 g/ml IPTG
Filter sterilize
Store aliquots at -20°C.
Lysis Buffer   5 mM DTT
15 mM KCl
Add DTT and Lysozyme just before use.
5 mM MgCl2
50 mM HEPES, pH 7.9
1 mM EDTA
10 mg/ml Lysozyme
Ampicillin Stock   Filter sterilize
100 mg/ml Ampicillin
LB Medium with Ampicillin   5 g/liter NaCl
10 g/liter Tryptone
5 g/liter Yeast Extract
1 ml/liter 1.0 M NaOH
Autoclave
When cool, add Ampicillin to the appropriate concentration
 
BioReagents and Chemicals
Preswollen Glutathione-agarose
IPTG
Ampicillin
Tris
Potassium Chloride
Sodium Chloride
HEPES
Magnesium Chloride
Sodium Hydroxide
Potassium Phosphate, Monobasic
DTT
Yeast Extract
Tryptone
Sodium Phosphate, Dibasic
EDTA
Glutathione
 
Protocol Hints
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