Denaturing purification of insoluble bacterial GST-fusion Proteins
1. Grow cells in 5ml (or more) of LB-Amp overnight for a starter culture.
2. In AM, seed 500-1000 ml larger culture using the overnight culture as a seeding culture. The culture is grown in LB-Amp medium. Aerate well with shaking at 37o C until OD at 600nm is ~0.6 (usually about 4 hrs).
3. Add the appropriate amount of IPTG stock solution to the culture to obtain a final IPTG concentration of ~2mM. (Add 4mls water to an unopened bottle= 1M. Induce 1L culture with 2mls.)
4. Continue shaking for 2-4 hours.
5. Spin down cells. Pellet may be frozen at -70 C overnight; this actually helps lyse the bacteria.
6. Resuspend pellet in 25 ml of lysis buffer #1-- PBS-CMF, 2mM EDTA, 10mM DTT, PMSF, benzamidine, aprotinin, leupeptin.
7. Cells are lysed by sonicating 3x15 seconds at 4o C.
8. Centrifuge at 20 - 30 K rpm in ultracentrifuge or in superspeed centrifuge at 15 krpm; 4o C for 30 min.
9. Pour supernatant into seperate container. Resuspend pellet by sonicating in 25 ml PBS with 10 mM DTT, 0.1% SDS, 10% glycerol. Recentrifuge.
10. Pour supernatant into seperate container. Resuspend pellet by sonicating in 25 ml PBS with 10 mM DTT, 2 M urea. Recentrifuge.
11. Pour supernatant into seperate container. Resuspend pellet by sonicating in 25 ml PBS with 10 mM DTT, 8 M urea. Recentrifuge.
12. (At this step you can check to ensure your fusion protein is in the 8 M supernatant by checking all of the supernatants on the gel).
13. Dialyse the 8 M supernatant sequentially against 6 M, 5 M, 4 M, 3 M, and 2 M urea in PBS with 10 mM DTT. Dialyze in small volume and alter the buffer no more often than every 2 hours. Some protein may crash between 2-3 M urea. Spin this out at high speed.
14. Wash 1-1.5 mls packed GSH-sepharose into 2M urea buffer, add to the dialysate. Rotate the filtered supernatant/sepharose at 4oC for 30-60min.
11. Spin, remove liquid and wash the sepharose 2x with PBS with 10 mM DTT and 2 M urea. Gel check for protein bound.
12. Transfer the washed beads into a small Dispo-column and wash with a few ml of 2 M urea buffer. Elute the fusion protein from the sepharose with 0.5 ml aliquots of 100 mM glutathione in 2 M urea buffer. Protein assay and gel check.
13. Dialyze fusion protein against 50mM Tris pH8.0 or PBS. The protein will likely crash, it is still good for anitgen. Freeze at -70 C.