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Denaturing purification of insoluble bacterial GST-fusion Proteins

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.