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MOLECULAR BIOLOGY: WORKING WITH DNA

EXPRESSION: FUSION PROTEINS

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

Purification of GST-Fusion Proteins from E. coli-Basic Protocol
Contributor: The Laboratory of Donald Rio at the University of California, Berkeley
 
Overview
This protocol is a general outline for the purification of GST-fusion proteins expressed in E. coli. After cloning the gene into the expression vector pGEX-sTK, check two to five clones for expression of the fusion protein at both 30°C and 37°C. Sometimes, as few as one in three clones tested express the fusion protein. One method for purifying a heterologously expressed protein of interest is to clone the gene in frame with the Glutathione S-Transferase (GST) coding sequence and introduce the resulting construct into E. coli. The GST-fusion protein is purified from a bacterial lysate by binding to an glutathione resin. After washing the resin of bacterial impurities, the fusion protein is eluted from the resin with reduced glutathione.
 
Procedure
A. Cell preparation (see Hint #2)

1. Inoculate 500 ml of Rich Media with 5 ml of an overnight culture of strain expressing the GST-fusion protein. Grow at the appropriate temperature to an optical density of 0.5 at 600 nm (OD600 of 0.5).

2. Optional: Collect 1 ml of uninduced cells and centrifuge in a microcentrifuge for 2 min. Discard the supernatant and resuspend by vortexing in 50 μl of 1X SDS-PAGE Sample Buffer. Store on ice.

3. Induce the bacteria with 1.5 ml of 0.1 M IPTG for 2 hr at 30°C (see Hint #3).

4. Optional: Collect 0.5 ml sample of induced cells and centrifuge in a microcentrifuge for 2 min. Discard the supernatant and resuspend by vortexing in 100 μl 1X SDS-PAGE Sample Buffer. Store on ice.

5.Centrifuge the culture for 10 min in a GSA rotor at 6,000 rpm (5,900 X g) to pellet the cells.

6. Remove the supernatant and resuspend the cell pellet in 10 ml of ice-cold Lysis Buffer.

7. Transfer the cell suspension to Ti70 tubes. Snap-freeze the cells in Liquid Nitrogen or a dry ice-Ethanol bath; or store the cells at -20°C until you are ready to proceed.

8. Thaw the frozen cells in cold water.

9. Sonicate with 15 second bursts at a setting of 4 with the tip of a cell sonicator probe. Minimize the foaming, keep sample in an ice-water bath during sonication, and cool on ice between each burst. Repeat sonication bursts until protein is no longer released. Monitor protein release of 10 μl aliquots of the lysate in the Bradford Assay (see Protocol ID#493). The suspension should be partially clear when finished.

10. Centrifuge at 4°C for 30 min at 9,000 X g.

11. Transfer the supernatant to a 15 ml conical tube. The crude extract can be frozen in liquid nitrogen or added to glutathione agarose beads (see Section C).

12. Optional: Resuspend the pellet in 10 ml of Lysis Buffer. Take a 5 μl aliquot of the resuspended pellet and add 5 μl of 2X SDS-PAGE Sample Buffer. Store on ice. Take 5 μl of the crude extract and add 5 μl of 2X SDS-PAGE Sample Buffer. Store on ice.

B. Glutathione-Agarose Bead Preparation

1. Weigh out enough Glutathione-Agarose to bind all of the fusion protein. 0.25 g of dry Glutathione-Agarose beads results in 3 ml of swelled bed volume and the capacity of the beads is 3 to 5 mg protein/ml Glutathione-Agarose. Place the powder in a 15 ml conical tube.

2. Add 10 ml of ddH2O and let the beads swell for 2 hr at room temperature.

3. Centrifuge in a table-top centrifuge at approximately 400 X g for 2 min to pellet the beads and remove the supernatant.

4. Resuspend the beads in 10 ml of ddH2O and centrifuge at approximately 400 X g for 2 min to pellet the beads and remove the supernatant.

5. Wash the beads twice with 10 ml of TE, centrifuging at approximately 400 X g for 2 min to pellet the beads, and remove the supernatant between washes.

6. Store the beads at 4°C in TE until use.

C. Fusion Protein Purification of Glutathione Agarose

1. Put the beads into either a 15 ml or 50 ml conical tube depending on the volume of your extract. Wash the beads twice in 10 ml of ice-cold Wash Buffer.

2. Centrifuge in a table-top centrifuge at approximately 400 X g for 2 min at 4°C to pellet the beads and remove the supernatant.

3. Add the cell extract to the beads and incubate the mixture with rotation at 4°C for 30 min. To keep the slurry at 4°C, place the tube in a 250 ml centrifuge tube filled with ice.

4. While the extract and beads are incubating, make up the Elution Buffer. It should be made fresh before each use.

5. Centrifuge the cell extract:Agarose slurry in a table-top centrifuge at approximately 400 X g for 2 min at 4°C to pellet the beads.

6. Place the supernatant into a conical tube. Take a 10 μl aliquot and add 10 μl of 2X SDS-PAGE Sample Buffer. Store the aliquot on ice. Freeze the supernatant in Liquid Nitrogen and store at -80°C.

7. Wash the pellet of Glutathione-Agarose beads three times in 10 ml of ice-cold Wash Buffer. It may be easier to transfer the beads to a 15 ml conical tube if a 50 ml conical tube was used for the binding step. Centrifuge the beads in a table-top centrifuge at approximately 400 X g for 2 min at 4°C between washings.

8. Add 0.5 to 1 volumes of cold Elution Buffer equivalent to the volume of beads.

9. Incubate the slurry with rotation at 4°C for 30 min.

10. Centrifuge the slurry in a table-top centrifuge at approximately 400 X g for 2 min at 4°C to pellet the beads and recover the supernatant.

11. Repeat the elution (Step #8 through #10) two or three more times and save the supernatants containing the eluted GST-fusion protein.

12. Optional: Take a 10 μl aliquot and add 10 μl of 2X SDS-PAGE Sample Buffer. Boil all the SDS-PAGE samples for 3 minutes. To analyze induction, yield, and purification of the fusion protein, load samples onto a 10% SDS-polyacrylamide gel (see Protocol ID#455). After the gel has run, stain with Coomassie Blue to visualize the proteins.

13. Freeze the eluate in Liquid Nitrogen and store it at -80°C (see Hint #4).

Solutions
0.1 M Isopropyl β-D-Thiogalactopyranoside (IPTG)   0.1 M IPTG (CAUTION! See Hint #1)
Sterile filter through 0.22 μm filter.
Protease Inhibitor Cocktail (1000X) (CAUTION! See Hint #1)   0.5 mg/ml Leupeptin
0.5 mg/ml Aprotinin
0.5 mg/ml Chymostatin
in ddH2O.
0.5 mg/ml Antipain
0.5 mg/ml Pepstatin
Elution Buffer   Filter through a 0.22 μm filter.
1 mM EGTA, pH 8.0
100 mM KCl
50 mM Tris-HCl, pH 8.0
0.5 mM DTT
20 mM reduced Glutathione (GSH)
Make this buffer just before each use.
1 mM PMSF
Confirm the pH, as GSH will make the buffer acidic. If necessary, adjust the pH to 8.0 with an appropriate base.
Wash Buffer   1 mM EGTA, pH 8.0
1 mM Phenylmethylsulfonyl Fluoride (PMSF) (CAUTION! See Hint #1)
Filter through a 0.22 μm filter before use.
0.05% (v/v) Igepal CA 630
100 mM KCl
50 mM Tris-HCl, pH 8.0
1 mM EDTA, pH 8.0
Add the DTT, PMSF, and the Protease Inhibitor Cocktail just before use.
1X Protease Inhibitor Cocktail
0.5 mM DTT
1 M NaCl
Rich Media   5 g/liter NaCl
5 g/liter Yeast Extract
When cooled, add Ampicillin to 100 μg/ml.
Autoclave
2 g/liter Glucose
10g/liter Tryptone
2X SDS-PAGE Sample Buffer   4% SDS
5% 2-Mercaptoethanol
0.005% Bromophenol Blue
20% Glycerol
125 mM Tris-Cl, pH 6.8
TE   10 mM Tris
pH 8.0
1 mM EDTA
0.1 M Isopropyl β-D-Thiogalactopyranoside (IPTG)
 
BioReagents and Chemicals
EGTA
IPTG
Ampicillin
Chymostatin
Pepstatin
Yeast Extract
Tryptone
Antipain
Dry Ice
EDTA
Liquid nitrogen
Aprotinin
Tris-HCl
Bromophenol Blue
SDS
Potassium Chloride
Sodium Chloride
Glutathione
Glucose
2-Mercaptoethanol
Igepal CA 630
Glycerol
Leupeptin
PMSF
DTT
 
Protocol Hints
1. This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions.

2. If expression or yield is poor in the host bacterial strain, try introducing the fusion plasmid into a protease deficient lon- and/or rpoH- host strain. lon- strains lack the activity responsible for degrading aberrant proteins. rpoH- strains lack a heat shock transcription factor that activates some stress-induced proteases. CAG629 is both lon- and rpoH-, but is also a difficult strain to work with. ER2508 and CAG597 are good alternatives; ER2508 is lon- and CAG597 is rpoH-. The CAG strains are difficult to transform by heat shock of competent cells. Often, the strains must be transformed by electroporation. These strains are available from New England Biolabs.

3. The induction can be carried out at 37°C; however, lower temperatures favor increased fusion protein stability. Incubations of 1 hour or 3 hours might obtain higher yields.

4. Approximately 50% of the protein remains bound to the beads even after a final elution overnight at 4°C.

 
Citation and/or Web Resources
1. Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 1988; 67:31-40