Sauer:ClpP purification/Untagged ClpP

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< Sauer:ClpP purification
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Buffers

Buffer L: 50 mM Tris-HCl (pH 7.6), 1 mM DTT, 0.5 mM EDTA, 10% Glycerol

Buffer L150: Buffer L + 150 mM KCl

Buffer L400: Buffer L + 400 mM KCl

Buffer L100: Buffer L + 100 mM KCl

Protocol

  • 1. Suspend cells expressing ClpP in 10mL/L culture of Buffer L150.
  • 2. Freeze at -80°C until use.
  • 3. Thaw by addition of 10mL/L culture Buffer L150.
  • 4. Add 1 mg/mL lysozyme, and let sit in ice, ~1 hour.
  • 5. Sonicate to reduce viscosity. (or use DNase or Benzonase, they need Mg++ and Ca++)
  • 6. Spin at 15K rpm in a SA-600 rotor, 30’.
  • 7. Add AmSO4 to 30% saturation (0.164 g/mL) while gently stirring on ice and incubate on ice for 30’.
  • 8. Spin at 10K rpm, 15’; save supernatant.
  • 9. Increase the AmSO4 to 60% saturation (0.181 g/mL additional) while stirring on ice and incubate on ice 30’.
  • 10. Spin at 10K rpm, 15’; save pellet.
  • 11. Resuspend pellet in Buffer L150 (I used 5 mL per 1L culture but this was more than I needed:Marylee).
  • 11a. Alternately, you can resuspend the pellet in "saltless" buffer. At 60% saturation, there is ~2.7 M AmSO4, so the remainder in the pellet volume contibutes plenty of salt. Resuspending in 20-30 pellet volumes will allow you to go directly to the anion exchange column without wasting PD-10 columns. If you do this, spin the resuspension to clear it if insoluble material before the anion exchange step.
  • 12. Desalt into a PD-10 column (with fresh Buffer L150) or decrease conductivity by dilution into L150.
  • 13. HiLoad 16/10 Q Sepharose HP separation. The mobile phase contains Buffer L150 and elution proceeds with a 200 mL linear gradient between 150mM and 400 mM KCl.
  • 14. Concentrate sample by precipitation in 60% AmSO4 (0.361 g/mL), and then incubate on ice for 30’.
  • 15. Spin at 10K rpm and resuspend in Buffer L100.
  • 16. Gel filtration using a HiPrep Sephacryl S-300HR column in Buffer L100.
  • 17. Concentrate fractions on a HiLoad 16/10 Q Sepharose column or by a spin concentrator.

Notes

To Avoid sonication, you can use DNase or Benzonase to reduce viscosity. Change the lysis buffer to contain Mg++ and Ca++ if you do. After treatment, add EDTA to chelate the metal and continue with the prep.

You can make a "5X" stock buffer: 125 mM HEPES-Tris, 2.5 mM EDTA, pH 7.6. Buffers are stronger and less sensitive to dilution if they are composed of paired buffers with pKas flanking the desired pH. HEPES ~7.4 and Tris ~8.1. Start with the buffer with a pK closest to your desired pH (125 mM HEPES in this case) and add Tris base until the pH is 7.6.

Make a 1 M KCl solution with 1/50th vol. of the 5X buffer above and adjust the pH to 7.6. Then making your 100, 150, and 400 mM buffers is a matter of mixing.

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