GST fusion protein purification

(from Smith (1988) Gene 67: 31.)

1. Need to get fusion plasmid into BL21 bugs; we usually use BL21(DE3)pLysS from Stratagene which give high expression and no toxicity to the bugs.

2. Grow a starter O/N culture of 50mL LB plus 50ug/mL amp.

3. Next am, dilute the O/N culture into a 500mL-1L flask of prewarmed LBAmp.

4. Shake at 37°C for 2-3 hours, until the O.D.600 = 0.4 to 0.5.

5. Add IPTG to 0.1-0.5mM. You may want to test the optimal level of IPTG for induction first by growing small 2mL cultures and trying induction with different amounts of IPTG. Then run 20uL on a gel for Coomassie staining - you should be able to see the band. You may also want to do this kind of a test to establish the optimal time for growing the cells in step #6.

6. Shake 37°C for 2 hours.

7. Save an aliquot to test on a gel for protein synthesized. Spin bugs down in Sorvall GS3 rotor at 5000RPM for 10'.

8. Resuspend in 20-50mL ice cold lysis buffer (PBS with 100mM EDTA, 1% Tx-100, 1% aprotinin, 1mM PMSF, 1mM DTT).

9. Sonicate on ice with probe sonicator (cold room) 3x1' at setting 5 (microtip maximum).

10. Spin to remove debris, GSA 5000RPM for 10'. Keep an aliquot of the pellet to run on the final gel, as sometimes GST-fusions may be insoluble and wind up in the pellet.

11. Can also filter through .45um filter to remove any left over particles.

12. Add glutathione agarose, 1-3mL/ liter original culture. To prepare the glutathione agarose Swell the powder in ddH20 (200mL/g) for 30' to O/N at RT. Spin down 3' in clinical centrifuge and wash several times with ddH20 to remove the lactose. Then bring up in the lysis buffer to equilibrate.

13. Rotate at 4°C for 1hour.

14. Wash 3X with ice cold PBS-100mM EDTA. Add 5-10mL wash solution, rotate for 1-2 minutes at 4°C and then spin in clinical centrifuge for 3' to pellet beads.

15. To elute protein from the beads, add 20mM reduced glutathione in 100mM Tris pH8.0. Rotate 15', then spin out beads.

16. To change the buffer of the fusion protein, pour over a PD10 column (Pharmacia) or dialyze against 1L of desired buffer O/N in a Slidalyzer cassette (Pierce).

17. To cleave the protein off of the GST ....HM...