Sauer:ClpA purification

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Contents

ClpA Purifcation Protocol

Jon Kenniston / Julia Flynn / John Lo


General Notes

  • Do all purification steps at 4°C.
  • Thoroughly wash columns before use.
  • All buffers should be filtered, degassed, and stored at 4oC.
  • Run fractions on 8% polyacrylamide gels (ClpA = 84 kD)
  • ClpA pI = 5.868 (Colibri), 6.3 (Maurizi)


Procedure

1. Grow O/N in 3 mL LB/Kan ClpA frozen stock (M169T in pET-9a, BL21(DE3)).

2. Inoculate 1 L LB/Kan each culture – 4 L total.

3. Grown until A600 1.0, then induce with 0.4 mM IPTG for 2 hours.

4. Spin at 4000 rpm for 20 minutes.

5. Resuspend pellets in 5 mL Lysis Buffer. Try adding salt to lysis buffer? 100 mM KCl sounds good here.

6. Freeze overnight in -80oC.

7. Thaw cells with additional 25 mL Lysis Buffer + Calbiochem protease inhibitor cocktail.

8. French Press 2X. (or sonicate, 5x 30s)

9. Spin at 19,000 rpm for 1 hour in Sorvall. Remove and keep supernatant.

10. Add 40 mL 100% saturated AmSO4 to the supernatant (~60 mL) for final concentration of 40%. Incubate at 4oC for 1 hour. (or overnight)

11. Spin at 12,000 rpm (20k x g) for 30 min. and resuspend pellets in 40 mL of S-column Buffer A (~6.25 mL/g).

12. Spin cloudy resuspension another 30 minutes at 12,000 rpm. Keep supernatant.

13. Check conductivity. Ensure that sample has less conductivity than S-column Buffer A alone. If needed, dilute or dialyze sample with S-Sepharose buffer without KCl, or add KCl if greatly under-conductive. (always overconductive. Dilute with S-Sepharose Buffer A, no salt)

14. Run sample over S-Sepharose column (<50 mL) and elute with gradient of 0.2 M – 1 M KCl in Buffer B at 0.5-0.75 mL/min.

15. Check fractions on SDS-PAGE and compare with original over-expression sample.

16. Combine fractions and add AmSO4 to be 0.6 M (~15%).

17. Equilibrate Phenyl-Sepharose column (10-20 mL) with Phenyl-Sepharose Buffer A with 0.6% CHAPS.

18. Load sample onto column.

19. Gradient Buffer A to Buffer B at 2 mL/min for 60 minutes

20. Pool peak fractions and concentrate using Amicon tubes 5,000 rpm.

21. Dialyze against 4 L HO buffer overnight. Change 2 to 3 times.

22. Make 100 µL and 50 µL aliquots, and store at -80oC.


Buffers

Lysis Buffer:

  • 50 mM Tris-Cl, pH 7.5
  • 2 mM DTT
  • 2 mM EDTA
  • 10% glycerol

S-Sepharose Buffer A:

25 mM HEPES, pH 7.5 200 mM KCl 2 mM DTT 0.1 mM EDTA 10% glycerol

S-Sepharose Buffer B:

  • 25 mM HEPES, pH 7.5
  • 1.0 M KCl
  • 2 mM DTT
  • 0.1 mM EDTA
  • 10% glycerol

Phenyl-Sepharose Buffer A:

  • 50 mM NaPO4, pH 7.5
  • 2 mM DTT
  • 10% glycerol
  • 0.6 M (NH4)2SO4

Phenyl-Sepharose Buffer B:

  • 50 mM NaPO4, pH 7.5
  • 2 mM DTT
  • 10% glycerol
  • 0.6% CHAPS

Dialysis (HO) Buffer:

  • 50 mM Tris-Cl, pH 7.5
  • 100 mM KCl
  • 1 mM EDTA
  • 10% glycerol
  • 2 mM DTT

4x ClpA Activity Buffer

  • 200 mM HEPES pH 7.5
  • 80 mM MgCl2 6H20
  • 1.2 M NaCl
  • 40% glycerol
  • 2 mM DTT
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