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MOLECULAR BIOLOGY: WORKING WITH DNA

EXPRESSION: FUSION PROTEINS

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

Purification of GST-Fusion Proteins from E. coli-Alternate Protocol
Contributor: The Laboratory of Andrew Murray at Harvard University
 
Procedure
1. Inoculate 6 liters of NZYCM media containing 75 μg/ml ampicillin with between 100 to 200 ml of an E. Coli culture grown overnight.

2. Grow cultures at 37°C until the optical density reaches 0.6 absorbance units at 550 nanometers.

3. Add IPTG to 0.1 mM and continue incubating the cultures at 30 to 37°C for another 2 to 3 hours.

4. Centrifuge the cells to pellet (usually 1,500 X g) in a clinical centrifuge and discard the supernatant.

5. On ice, resuspend the pellet in a small volume of PBS and keep on ice.

6. Combine the resuspended PBS cell pellets from as many cell culture preparations as needed to obtain enough fusion protein.

7. Centrifuge the cell suspensions to pellet the cells (usually 1,500 X g) in a clinical centrifuge and discard the supernatant.

8. Freeze the cell pellet in liquid nitrogen.

9. Resuspend the frozen pellet in approximately 5 volumes of PBS Incubation Solution using constant stirring (i.e. stir bar and magnetic stir plate).

10. OPTIONAL: Sonicate cells briefly using a sonicating water bath (see hint #1).

Remaining steps must be performed at 4°C.

11. Add KCl to 0.25 M final concentration and DTT to 15 mM final concentration.

12. Centrifuge the lysate for 60 min at 111,500 X g at 4°C (35,000 rpm using a Beckman Type 50.2Ti rotor).

13. Save the supernatant.

14. Load supernatant, over a period of approximately 2 to 4 hours, onto a 10 ml glutathione agarose column (prepared according to manufacturers instructions) .

15. Wash the column with 50 to 100 ml of PBS Column Buffer.

16. Determine the protein concentration from the last volume of PBS Column Buffer wash from the column (see protein concentration by Bradford protein).

17. If there is a detectable level of protein in the final wash fraction from the glutathione agarose column then continue washing the column with PBS Column Buffer until there is no detectable protein.

18. Elute the column with Elution Buffer by adding 1/7th column volume to the top of the column and collecting the 1/7th volume fraction.

19. Assay each 1/7th column volume fraction for protein concentration (see protein concentration by Bradford protein).

20. Pool the peak fractions.

21. Dialyze pooled peak fractions extensively (overnight or even longer) at 4°C against Dialysis Buffer.

22. Yield of GST is usually around 100 to 200 mg while the yield of GST-fusion protein is generally lower.

Solutions
Dialysis Buffer   30% (v/v) Glycerol
50 mM HEPES, pH 7.6
50 mM KCl
Elution Buffer   0.25 M KCl
50 mM Tris, pH 8.1
*Add just before use.
5 mM Reduced Glutathione*
PBS Column Buffer   0.25 M KCl
*Add just before use
0.5 mM DTT*
Prepared in PBS
150 mM DTT   Prepared just before use.
250 mM KCl
PBS Incubation Solution   200 μg/ml Lysozyme
*Add just before use
1 mM EDTA
Prepared in PBS
1 mM PMSF* (CAUTION See Hint #2)
PBS   pH 7.2
138 mM NaCl
140 mM Sodium Phosphate Dibasic (Na2HPO4)
2.7 mM KCl
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
5 M NaOH
NZYCM Media   Combine ingredients in 950 ml distilled deionized water (ddH2O).
5 g NaCl
Bring the volume to 1 liter using ddH2O.
5 g Bacto-Yeast Extract
Sterilize by autoclaving for 20 min on liquid cycle.
Adjust pH to 7.0 using 5 M NaOH
1 g Casamino Acids
Shake until the solutes have dissolved
10 g NZ Amine (Casein Hydrolysate Enzymatic)
2 g MgSO4
 
BioReagents and Chemicals
Reduced Glutathione
Glycerol
PMSF
Magnesium Sulfate
EGTA
Ice
Lysozyme
Tris
Potassium Chloride
Sodium Chloride
DTT
Casamino Acids
Bacto-Yeast Extract
IPTG
Nitrogen, Liquid
Ampicillin
HEPES
Potassium Phosphate, Monobasic
Sodium Hydroxide
Sodium Phosphate, Dibasic
KCl
NZ Amine (Casein Hydrolysate Enzymatic)
 
Protocol Hints
1. The optional step is to ensure that cell walls have been lysed.

2. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.