Sauer:ClpX purification/Protocol 1

From OpenWetWare
Revision as of 18:49, 25 July 2006 by Kathmc (talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

Home        Protocols        Lab Members        Materials        Equipment        Links        Internal       

Protocol 1

Cell growth

ClpX can be expressed in a variety of cell types, but greater success usually results from strains that do not have "leaky" expression. Tight control of expression can usually be obtained with a BL21 pLysS system, but others work as well.

1. Grow cells to a high density (~A600=1.0) at 37 ˚C in either LB or TB media. Then, shift temperature to 25 ˚C. Shortly thereafter, induce ClpX with 0.5–1 mM IPTG for 3 hr.

2. Harvest cells by centrifugation. Resuspend in 1 mL lysis buffer (see buffer recipes below) per gram of wet cell pellet. The paste can be stored at -80 ˚C until you are ready to purify ClpX.

Lysis/ammonium sulfate((NH4)2SO4) precipitation

1. Thaw cells and add 10 mL lysis buffer per gram of cell pellet. Disrupt cells by either French press or gentle sonication.

2. Spin at 13–15K rpm in Sorvall RC5B centrifuge with SA600 rotor for 1 hr to pellet large cell debris.

3. Transfer supernatant to appropriate tubes and spin in ultracentrafuge (Baker lab) in Beckman Ti45 rotor for 1 hr at 40K rpm.

4. Transfer the supernatent to new tubes. Add (NH4)2SO4 to 35% (saturation not w/v) and stir at 4 ˚C for 1 hr–overnight.

5. Spin at slow speed (4K in Beckman J6-HC swinging bucket rotor) to pellet (NH4)2SO4 precipitate. If you spin too fast here, resuspension is more difficult.

6. Just in case, take supernatent and add NH4SO4 to 45% and stir at 4 ˚C. Spin as in step 5.

7. Resuspend the (NH4)2SO4 pellet in lysis buffer (use minimal volume so you have a reasonable load volume for the phenyl sepharose column). After allowing as much of the precipitate as possible to resolubilize (some material typically remains insoluble), spin the sample for 1 hr at 12K in Sorvall RC5B centrifuge with the SA600 rotor. Keep the supernatant.

8. Run samples of lysis supernatant and pellet, and (NH4)2SO4 supernatants and pellets on an SDS-polyacrylamide gel to make sure you haven't left all the ClpX behind.

Phenyl sepharose column

1. Add (NH4)2SO4 to the ClpX sample until the conductivity matches the conductivity of the phenyl sepharose equilibration buffer (buffer PS-A). Obviously, too much (NH4)2SO4 at this step will precipitate the ClpX.

2. Equilibrate a HiLoad 16/10 phenyl sepharose high performance column (Amersham) with buffer PS-A, load sample, and wash column with buffer PS-A.

3. Run gradient from 0–100% buffer PS-B. ClpX generally elutes half way through the gradient.

4. Run a gel of the fractions and combine those that contain ClpX.

Q sepharose column

1. Buffer exchange combined phenyl sepharose fractions containing ClpX into buffer QS-A (dialyze, do not use a PD-10 column; ClpX sticks to PD-10s). Alternatively, dilute the ClpX sample to a conductivity equivalent to that of buffer QS-A.

  • I took the dilution route and ended up with ~500 mL of sample, which I loaded onto the column using the peristaltic pump. It took more than 2 hr to load the sample this way, but I programmed to pump to stop automatically so I could leave and come back. This procedure may or may not save you time versus an overnight dialysis step. Kathleen

2. Equilibrate a HiLoad 16/10 Q sepharose high performance column (Amersham) with buffer QS-A, load sample, and wash column with buffer QS-A.

3. Run gradient from 0–100% buffer QS-B. ClpX generally elutes around 300 mM KCl.

4. Run gel and pool fractions that contain ClpX. Determine the concentration by UV absorbance (extinction coefficient @280 nm=84480 cm-1M-1 for ClpX6). Aliquot and keep samples frozen at -80 ˚C. Some recommend only thawing a tube once, as multiple freeze/thaw cycles results in loss of activity.

Source 15Q column

To concentrate the ClpX sample, run a smaller volume Source 15Q column (Amersham) with a steep gradient using the same buffers you used for the Q sepharose column. ClpX typically sticks to spin columns used for concentration, so you will lose protein if you try to concentrate your sample this way.

More columns...

If you are unhappy with the purification at this point, you can try running a gel filtration column (S200, Amersham) and/or a hydroxylapatite column.


Lysis buffer

50 mM Tris-HCl, pH 8.0

100 mM KCl

5 mM MgCl2

5 mM DTT

10% glycerol

1 mM PMSF (make fresh!)

Bufer PS-A

50 mM Na-phosphate buffer, pH 7.5

2 mM DTT

10% glycerol

0.5 M (NH4)2SO4

Buffer PS-B

Same as PS-A, but with no (NH4)2SO4.

Buffer QS-A

Same as lysis buffer, but with no PMSF.

Buffer QS-B

Same as QS-A, but with 1 M KCl (or you could use NaCl here).