Materials and Reagents

Escherichia coli (E. coli) strain of choice
IPTG (Sigma-Aldrich)
Imidazaole (Sigma-Aldrich)
Protease inhibitor (Roche Diagnostics)
Lysozyme (Sigma-Aldrich)
Glutothion sepharose 4B beads (Thermo Fisher Scientific/Pierce Antibodies, catalog number: 15160 )
Glutathione (Thermo Fisher Scientific/Pierce Antibodies, catalog number: 78259 )
NP-40 (Sigma-Aldrich)
Bacto trypton
Yeast extract
Liquid nitrogen
NaCl
KCl
Na₂HPO₄
KH₂PO₄
Protease inhibitor
Lysis buffer (see Recipes)
1x PBS buffer (see Recipes)
Elution buffer (see Recipes)
2x YT medium (see Recipes)

Equipment

Sonicator
Centrifuges
Pellete
Water bath

Procedure

Small scale expression and purification test
1. Grow 5 ml E. coli culture harboring the expression construct in 2x YT medium overnight (12-16 h) at 37 °C.
2. The next day, 1: 20 dilute the overnight E. coli culture into fresh 2x YT medium. Grow at 37 °C to OD₆₀₀ of 0.6-0.8. Depending on E.coli strain and status, the time should be around 2-3 h.
3. Induce the culture with 0.1 mM IPTG. Take 10 ml culture out at 0 h, 1 h, 2 h, 4 h and overnight, spin down and flash freeze in liquid nitrogen, then stored in -80 °C.
4. Once all the samples are collected, take the pellete, resuspend in 1.5 ml lysis buffer. Incubate on ice for 30 min.
5. Sonicate each sample on ice for 15 sec for twice.
6. Centrifuge the lysate at top speed at 4 °C for 30 min. Separate the supernatant and pelletes. Resuspend the pelletes in 1.5 ml lysis buffer. Take 10 μl from supernatant and pellete suspension for sampling.
7. Add the 10 L glutothion sepharose 4B beads, incubate at 4 °C for 1 h and wash with 200 μl 1x PBS three times. Collect the supernatant after spinning down the beads for sampling.
8. Elute the beads with 10 μl elution buffer.
9. Run the samples from each step in appropriate SDS-PAGE gel to determine the expression and purification condition, and protein expression amount.
  Note: If there is solubility issue, try decrease the IPTG concentration and expression temperature.
Large scale expression and purification
1. Grow 50 ml E.coli overnight culture at 37 °C.
2. Dilute the over night culture to 1 L culture and grow at 37 °C till OD₆₀₀=0.4-0.8.
3. Induce with 0.1 mM IPTG and grow at 37 °C for appropriate amount of time.
4. Harvest the cells at 4,000 x g for 10 min at 4 °C. Flash freeze the pellete in liquid N₂ and store at -80 °C.
5. Thaw the cells in room temperature (RT) water bath. Put in ice and resuspend in 5-7x pellete volume of lysis buffer supplemented with protease inhibitor and lysozyme (See small scale expression and purification test). Incubate on ice for 30 min.
6. Sonicate on ice for 15 sec for 3 times, until the viscosity decrease. Be careful not let the lysate temperature goes beyond 10 °C. Save 10 μl for sample.
7. Centrifuge the lysate at 40,000 x g for 30 min at 4 °C. Discard the pellete. Save samples from supernatant and pelletes.
8. According to the estimated GST protein amount from small scale expression and purification test and binding capacity of glutothione beads in the manual, add appropriate amount of beads. To ensure purity, the amount of express proteins should be in excess of beads binding capacity.
9. Add 1% NP-40 to reduce the unspecific binding. Incubate at 4 °C for 30 min.
10. Transfer the slurry into a disposable column and collect the flow through.
11. Wash the beads with 20x beads volume PBS. Collect the flow through.
12. Add 1x beads volume of elution buffer. Collect the elution.
13. Repeat step 12 for 5 times.
14. After elution, take small amount of beads, add SDS loading buffer for sample.
15. Run the samples collected from lysate, pellete, supernatant, flow through, wash, elute and beads in SDS-PAGE gel.
16. Collect the elution containing your GST-proteins and dialysis against PBS at 4 °C for at least 1 h for three times.
17. Flash freeze at liquid nitrogen and store at -80 °C.

Recipes

2x YT medium
Bacto trypton
16 g
Yeast extract
5 g
NaCl
5 g
H₂O
800 ml
Adjust pH to 7.2
Add H₂O to 1 L
Autoclaved
PBS buffer (pH 7.4) (1 L)
NaCl
8 g
KCl
0.2 g
Na₂HPO₄
1.44 g
KH₂PO₄
0.24 g
H₂O
800 ml
Adjust pH to 7.4.
Adjust volume to 1 L with additional distilled H₂O
Sterilize by autoclaving
Lysis buffer
50 mM
NaH₂PO₄ (pH 8.0)
300 mM
NaCl
10 mM
Imidzaole,
1x Protease inhibitor
1 mg ml^-1 lysozyme
Elution buffer
20 mM
glutathione
75 mM
Tris (pH 8.0)
150 mM
NaCl

Acknowledgments

This protocol was adapted from Reference 1. Funding from the NIH supported this work.

References

Pierce Glutathione Agarose Instructions booklet: http://www.piercenet.com/instructions/2162248.pdf.