Sauer:ClpP purification/Untagged ClpP

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&deg;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.