GST protein purification from bacteria

GST purification from bacteria

Purification of GST-tagged protein
Daniel Mordes 11-12-06
Used for TopBP1 proteins

Note: I (Dave) recommend the following changes to the protocol below:

1. Sonicate a bit less, perhaps 3x 15sec.
2. Increase the speed of the centrifugation to clear the lysate to 15,000xg for 10min.
3. Consider binding at room temperature and be sure to check the lysate after binding to beads to see if you captured efficiently. My experience is that some proteins require a long time to bind the beads and may not bind efficiently in this buffer at 4 degrees.
4. Increase the Tris concentration in the elution buffer to 50mM. Glutathione will change the pH so you need to be sure to have enough buffer.
5. Dialysis 2x 500ml. For ATR kinase assays use 10mM Hepes pH=7.5 and 50mM NaCl. I have also added 0.5% glycerol successfully and you may be able to increase the glycerol concentration to stabilize the protein.

This protocol is for a 200ml bacterial culture. It can be scaled up proportionally.

Production of protein

Use culture (BL21codon plus cells) that has been show to be inducible to inoculate 25ml overnight culture of LB-Kan (or Amp) + chloramphenicol

Next day:

Use 5ml of overnight culture to inoculate 200ml LB (no antibiotics)
Periodically check OD600.

Once OD600 reaches ~0.6 (about 2 hours), induce bacteria with 200ul of 1M IPTG to give a final concentration of 1mM IPTG. Note make 1M IPTG fresh each time.

Incubate cultures at 25oC for 4 hrs.

Pellet bacteria, spin at 5000g for 15 minutes at 4¬0C.
Remove supernatant and store pellet at -20oC until ready to purify protein.

Purification of protein

NET Buffer:

Additives (add fresh)

1mM DTT
0.1mM PMSF
Aprotinin
Leupeptin

Resuspend pellet in 15ml (7.5ml/ 100ml bacterial culture) pre-chilled NET buffer with additives.

Transfer to round bottom 40ml tube.

Sonicate suspension 3 times in cold room. Setting 4, Duty cycle 90, 20 seconds.
Incubate suspension on ice for 1 minute between sonications.

Add 780ul of 20% Triton X-100 to give a 1% final concentration.

Incubate on ice for 30 minutes.
Meanwhile, aliquot 200ul glutathione sepharose bead slurry in microcentrifuge tube. Wash beads twice with 1ml of NET buffer. For washes, centrifuge beads at 500g (.5 rcf) for 1 minute.

Centrifuge 5000g for 10 minutes at 4oC.

Transfer supernatant (cleared lysate) to new tube.

Take 20ul sample of cleared lysate and take 20ul sample of pellet (to run on gel_.
Store pellet at -20oC.

Incubate cleared lysate with washed glutathione beads on rotator in cold room for 2.5 hours.

Wash beads three times with 10 ml NET buffer containing additives and 1% TritonX.
Each wash: Incubate for 5 minutes on rotator in cold room at 4oC. Then centrifuge beads at 1000rpm for 3 minutes at 40C.

Meanwhile, prepare Elution buffer-
75mM Tris pH 8.0. 15 mM Glutathion. 0.1 ug/ul Leupeptin
1ml of buffer: 75ul of 1M Tris pH 8.0, 4.61mg Glutathione, 1ul Leupeptin stock

After washes, transfer beads to microfuge tube. Take 10ul sample of beads. Spin down beads (30sec, 1 rcf) and remove supernatant.

Elute protein from beads. Typically, perform 4 elutions.
Use 200ul elution buffer for 200ul bead slurry (equal volumes).
Each elution: Incubate on rotator for 5 minutes at Room Temp. Then centrifuge beads at 500g (.5 rcf) for 30-60 seconds. Store supernatant on ice. Keep each elution separate. Keep elutions on ice.

Take 20ul sample of each elution.

After elutions, take 10ul sample of beads. Store remaining beads on ice.

Run samples (8) on gel.
Cleared lysate, pellet, beads before elution, each elution sample (4), beads after elution.

Coomassie stain gel.

Combine desired elutions and procede with dialysis.