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Procedure for Ion (DEAE) Exchange Chromatography for ecT bR2 Purification
Posted on Friday, November 21, 2003
Description
Procedure for Ion (DEAE) Exchange Chromatography for ecT bR2 Purification
Procedure
For cells from a 3.0 liter culture:
1. Prepare 400ml of 50mM Imidazole/0.6MNaCl/pH 7.0
2. Next, set up one 45 ml bed volumecolumn of DEAE-Sepharose in a 2.5cm diameter column.
3. The resin is washed using 400 mlof the buffer prepared in step one. (Note: Skip steps oneand three if using new or regenerated resin.)
4. Next, the resin is washed with 600mlof 25mM Imidazole / pH 7.0
5. Now prepare the Urea containing buffers: 1.2L 8.0M Urea/25mM Imidazole/pH 7.0
6. Add protease inhibitors to a finalconcentration of: 1mM Benzamidine (prepared fresh in degassed H2O), 1mM Leupeptin (Stock solutionmay be aliquoted and frozen @ -20ˇăC), 500mM EDTA
7. The column is now equilibrated with150 ml of the Urea buffer prepared in step 5.
8. At this point, the cell pellet isresuspended in 180 ml of buffer from step 5.
9. The samples are then placed in sonicatorcups and sonicated on ice: 1cycle, where each cycle is 2.5min. process time, 50% duty cycle, power =8.5, macrotip.
9. Next, the cell debris is pelleted(JA-17 rotor 17k rpm, 15 min. 4ˇăC). The supernatants are combined in a bottle and the pH is checked andadjusted to 7.0 (to ensure binding to the column).
10. The supernatant is slowly loaded (3ml/min) to the Urea equilibrated column from step 6.
11. The column is then further washed with 200ml of theUrea containing buffer from step 5.
12. Set up gradient and run: 300ml 8.0M Urea/25mM Imidazole/250mM NaCl/protease inhibitors/pH7.0 and 300 ml 8.0M Urea/25mM Imidazole/protease inhibitors/pH 7.0. Collect 7.5ml fractions (ˇÖ80 tubes). Run 5 ml aliquots from every 5th fraction on a 12%SDS-PAGE. Store tubes at 4ˇăCuntil the gel is developed.
13. Once the fractions containing protein are identified,pool the samples into a low salt and a high salt pool and add solid DTT to50mM final concentration. Stirfor 30 min. DO NOT CONCENTRATE!
14. Dialyze 5 x 4 hrs. against 4 L of 20mM AcOH, using8K MWCO (boiled in 0.005M EDTA). (Place low salt and high salt pools in separate dialysis bags) Pools may be kept separate throughout the folding and purificationprocess.
15. At this point, the concentration of the protein isdetermined by recording the absorbance of a 1:10 dilution in H2O at 280nm. (Assume 1 A280 = 1 mg/ml)
16. Next, prepare enough folding solution (200mM Tris-AcOH/2mM reduced glutathione/0.5mM oxidized glutathione/pH 8.0) so that once the unfoldedprotein solution in HAc and folding solution are combined, the final proteinconcentration is ˇÜ 1.0 mg/ml. The actual folding is done by adding the unfolded protein solution in HAcdropwise to a stirring solution of folding buffer (at least an equal volume). Once the addition is complete,the solution is stirred gently for 12 hrs. at room temperature.
17. Once the folding is complete, the sample is concentrateddown to a volume of approximately 45ml using an Amicon stirred cell witha YM3 membrane.
18. Next, the sample is dialyzed 2 x 4 hrs. against 1L25mM MES pH 6.0 at 4ˇăC in 8K MWCO dialysis tubing.
19. The sample is then dialyzed 4 hrs. against 500 ml 25mMMES pH 6.0 containing the reversible inhibitors (1mM Benzamidine, 1mM Leupeptin, 1mM EDTA)
20. Add irreversible inhibitors to a final concentrationof: 1mM TLCK (prepare fresh10mM in 1mM HCl) and 1mM PMSF (prepare fresh 5mg/ml in isopropanol) and incubatefor 4 hrs.
21. Next, the sample is centrifuged (17k rpm/20min/4ˇăCJA-17 rotor) to remove any precipitate. The sample is then loaded through a 0.22m syringe filter onto the ˇ°superloopˇ± of the AKTA purifier.
22. The protein is then purified on a 10ml Resource Q ionexchange column by gradient elution from 0 ¨C 30%B over 10 column volumeswith Buffer A being 25mM MES pH6.0/1mM Benzamidine/0.5mM TLCK/0.5mM PMSF/1mMEDTA and Buffer B being BufferA + 0.5M NaCl. Typically 10 runs, each with 5.0ml injection are conducted. This can be automated using the scouting feature on the AKTA.
23. The fractions corresponding to monomeric ecTbR2 are pooled and the sample is then concentrated to approximately1ml using the 3ml Amicon concentrator with a YM 3 membrane (shiny side up).
24. The concentration of protein is then determined byrecording the absorbance of a 1:100 dilution in H2O at 280nm andthe sample stored at ¨C20ˇăC.
25. Check for proteolysis by incubating a sample at 37ˇăCfor 24 hrs. and running SDS-PAGE
26. If proteolysis is detected, add TLCK and PMSF directlyto a final concentration of 0.5mM.
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