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MOLECULAR BIOLOGY: WORKING WITH DNA

EXPRESSION: FUSION PROTEINS

Purification of GST-Fusion Proteins from E. coli

Purification of GST-Fusion Proteins from E. coli
Contributor: The Laboratory of Jasper Rine at the University of California, Berkeley
 
Procedure
A. Initial Test to Check for Expression

1. Transform construct into competent E. coli. and pick four transformants.

2. Inoculate 2 ml cultures and grow at 37°C until the OD600 reaches 0.5.

3. Set aside 1 ml of each culture as uninduced controls. Add 1 μl of 0.4 M IPTG to the other 1 ml of the cultures.

4. Grow the cultures at 37°C for 3 hr.

5. Pellet the cells by pulsing in a microcentrifuge for 5 to 10 sec. Remove the supernatants and resuspend the cell pellets in 50 μl of ddH2O. Add 1 μl of PMSF to each yeast suspension and then add 50 μl hot 2 X SDS Sample Buffer (pre-warm the buffer to 100°C for 1 min before use). Mix the sample rapidly and boil for 3 to 5 min.

6. Place samples on ice (or freeze at -20°C, then thaw and boil for 1 min).

7. Centrifuge in a microcentrifuge for 30 sec.

8. Load 5 μl of the supernatant on a normal SDS-PAGE gel (see Protocol ID#455).

9. Stain the gel with Coomassie Blue (see Protocol ID#716)

B. Check the Solubility

1. If the appropriate protein is present in the stained gel in Step #9 above, then repeat Step #2 above by inoculating 100 ml cultures with E. coli.

2. Set aside 1 ml as an un-induced control as in Step #3, add 99 μl of 0.4 M IPTG to the remaining 99 ml of culture, and grow at 37°C for 3 hr.

3. Pellet the cells and resuspend the cells in 5 ml of PBS. Sonicate three times for 30 sec with a 30 sec incubation on ice between sonications. Remove a small sample from the lysate and centrifuge it at high speed to pellet any whole cells or debris. Remove the supernatant, add it to 2X SDS Sample Buffer, and boil for 3 to 5 min. Load 5 μl of the sample on an SDS-PAGE gel and look for the band of interest. If the band is present, the GST-fusion protein is soluble.

C. Purification of GST-Fusion Protein.

1. Add GST beads to the supernatant. Incubate for 30 min at room temperature with gentle stirring. Centrifuge the sample to pellet the beads and save the supernatant at 0°C. Resuspend the beads in PBS and re-pellet them. Repeat the PBS resuspension two more times.

2. Elute with Glutathione or Thrombin. Adding the Glutathione elutes the full-length fusion protein. Incubate overnight and pellet beads. Recover the fusion protein-containing supernatant.

Solutions
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
SDS Sample Buffer (2X)   0.002% (w/v) Bromophenol Blue
40% (v/v) Glycerol
8% (w/v) SDS
4% (w/v) 2-Mercaptoethanol or 6.2% (w/v) DTT
250 mM Tris
0.4 M IPTG
 
BioReagents and Chemicals
Thrombine
Sodium Chloride
Glutathione
Bromophenol Blue
SDS
Tris
IPTG
Potassium Phosphate, Monobasic
Sodium Phosphate, Dibasic
2-Mercaptoethanol
Glycerol
DTT
Potassium Chloride
 
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