TITLE: Expression	and	purification	of	PPM1H phosphatase
AUTHORS: Kerryn Berndsen, Axel Knebel, Pawel Lis, Amir Khan, Dario Alessi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A subset of small GTPases of the Rab family including Rab8A (Uniprot: P61006) and Rab10 (Uniprot: P61026) have been identified as substrates of the Leucine Rich Repeat Kinase 2 (LRRK2; Uniprot Q5S007)) (Steger et al., 2016). Recent work has revealed that Protein Phosphatase PPM1H (Uniprot Q9ULR3) is a cellular phosphatase that efficiently dephosphorylates LRRK2 phosphorylated Rab proteins (Berndsen et al., 2019). In order to study in detail the kinetics, mechanism and structure of PPM1H, it is necessary to produce milligrams of pure PPM1H protein. Here we describe in detail the method we use to produce milligram quantities PPM1H and several mutants.</div></div>

[STEPS]
?. [Transformation of plasmid into competent bacteria]
Mix  of pET15b 6HIS-SUMO-PPM1H plasmid (around ) with - of the competent BL21(DE3) cells and incubate  for .
10 µl
50 µl
100 µl
on ice
?. [Transformation of plasmid into competent bacteria]
Transfer the vial to a heat block equilibrated at  and leave for .
42 °C
?. [Transformation of plasmid into competent bacteria]
Transfer the vial back into ice and add  SOC medium and mix gently.
1 mL
?. [Transformation of plasmid into competent bacteria]
Incubate for  at  for recovery.
37 °C
?. [Transformation of plasmid into competent bacteria]
Plate  of the transformation onto a LB broth/ agar plate supplemented with  Carbenicillin.
0.1 mL
?. [Transformation of plasmid into competent bacteria]
Leave the plate  in a  incubator.
37 °C
?. [Overnight culture]
Using a  sterile pipette tip, remove one of the colonies from the plate and drop the tip into  LB broth medium, supplemented with  Carbenicillin.
200 µl
100 mL
50
?. [Overnight culture]
Incubate at  with  -  rotational shaking .
37 °C
Centrifuge: 180 33
Centrifuge: 200 33
The culture medium should become totally opaque in the morning.
?. [Set up cells and induce expression]
Decant 3 x  LB broth medium into 3 x  conical flasks.
1 L
2 L
?. [Set up cells and induce expression]
Supplement each flask / litre with  of  Carbenicillin.
1 mL
?. [Set up cells and induce expression]
Mix and add -  of the  culture into each flask.
10 mL
25 mL
?. [Set up cells and induce expression]
Incubate for  at , using an Infors Shaker-Incubator set at .
37 °C
Centrifuge: 200 33
?. [Set up cells and induce expression]
Sample two or three of the expressions by removing  medium and comparing the optical density at  with fresh LB medium, using a WPA cell densitometer or a spectrometer.
1 mL
600
?. [Set up cells and induce expression]
When the OD600 has reached 0.7-0.9, change the temperature setting of the Infors incubator to  and incubate the cells for at least another hour, all the while shaking at .
16 °C
Centrifuge: 200 33
?. [Set up cells and induce expression]
When the flasks have cooled down to  or lower, induce PPM1H expression by supplementing the medium with  IPTG.
20 °C
This is achieved by adding   of a   IPTG stock solution to each litre.
?. [Set up cells and induce expression]
Leave the cells to express the protein for  at .
16 °C
?. [Collection of cells and preparation of lysate]
The following morning prepare  of cell collection buffer and chill .
0.5 L
on ice
?. [Collection of cells and preparation of lysate]
Decant the content of the  conical flasks into  Beckman centrifuge pots, close the pots with their screw-cap lids and sediment the cells by centrifugation for  at  at  using a Beckman J6 centrifuge with the 6 x  rotor (4.2).
2 L
1 L
4 °C
Centrifuge: 4200 33
1 L
?. [Collection of cells and preparation of lysate]
Collect and open the pots and carefully decant the spent supernatant medium back into the flasks.
The flasks can now be sent for cleaning and autoclaving. The cell sediment in the pots is expected to have a volume of   –  .
?. [Collection of cells and preparation of lysate]
Add  of cold cell collection buffer to the sediment.
18 mL
?. [Collection of cells and preparation of lysate]
Transfer the  pots to the Infors incubator and set the temperature to  and the rotation to .
1 L
14 °C
Centrifuge: 110 33
?. [Collection of cells and preparation of lysate]
Leave the pots for , after which time the cell sediment should have completely resuspended.
?. [Collection of cells and preparation of lysate]
Pool all suspensions into one of the pots using a  pipette and a good pipettor. If any of the sediments has not well resuspended, pipette up and down close to the bottom of the pots.
25 mL
It is very useful at this stage to supplement the NaCl concentration to   and add glycerol to 5% (by vol) final concentration. It is important to add the NaCl and glycerol after the resuspension step and not before as this interferes with the resuspension.
?. [Collection of cells and preparation of lysate]
Once the NaCl and glycerol has been mixed in,  samples are aliquoted into  centrifuge vials and freeze them in liquid nitrogen for at least .
45 mL
50 mL
?. [Collection of cells and preparation of lysate]
Store the vials at.
-20 °C
The freezing and subsequent thawing step breaks up the cells and improves yield. Vials can be stored at -20ºC for up to 4 weeks.
?. [Preparation of cell lysate and pulldown of His-SUMO-PPM1H on Cobalt-resin]
Slowly thaw the vials with the cell suspension in cold water.
?. [Preparation of cell lysate and pulldown of His-SUMO-PPM1H on Cobalt-resin]
After thawing chill suspension  and then sonicate, using a probe sonicator (Cell disruptor). Settings: 6 – 8 pulses of  with  pauses. Set the amplitude to 50%.
on ice
?. [Preparation of cell lysate and pulldown of His-SUMO-PPM1H on Cobalt-resin]
Transfer the sonicated suspension into  Beckman centrifuge vials and sediment the insoluble material by centrifugation for  at  and  using a JA25.50 or a JA30.50 rotor in a Beckman Avanti centrifuge.
50 mL
Centrifuge: 40000 34
4 °C
?. [Preparation of cell lysate and pulldown of His-SUMO-PPM1H on Cobalt-resin]
Recover the supernatant by carefully decanting it into  conical centrifuge tubes. During the centrifugation step equilibrate  Cobalt-NTA-resin ( of a 50% slurry by vol, sufficient for a 3 L expression) by washing it three times with Milli Q water and once with cell collection buffer.
50 mL
3 mL
6 mL
?. [Preparation of cell lysate and pulldown of His-SUMO-PPM1H on Cobalt-resin]
Add a  of a 50% slurry (by vol) of the washed Cobalt-NTA-resin in collection buffer to each  vial with the lysate and incubate the mix on a Roller Mixer for  in a cold room set at .
1 mL
50 mL
4 °C
?. [Preparation of cell lysate and pulldown of His-SUMO-PPM1H on Cobalt-resin]
Avoid excessive agitation and especially formation of foam.
?. [Preparation of cell lysate and pulldown of His-SUMO-PPM1H on Cobalt-resin]
In the meanwhile, prepare the washbuffer.
?. [Preparation of cell lysate and pulldown of His-SUMO-PPM1H on Cobalt-resin]
Sediment the Cobalt-NTA-resin by centrifugation at  for  at  using a Eppendorf R5810.
Centrifuge: 1000 34
4 °C
?. [Preparation of cell lysate and pulldown of His-SUMO-PPM1H on Cobalt-resin]
Remove the lid and carefully remove the supernatant containing the depleted lysate using a  pipette, being careful not to disturb the resin.
10 mL
?. [Preparation of cell lysate and pulldown of His-SUMO-PPM1H on Cobalt-resin]
Add  of wash buffer. Prepare a  pipette tip by removing  –  from the pointed end using scissors. This allows it to be used to facilely resuspend the agarose.
3 mL
1000 µl
5
7
?. [Preparation of cell lysate and pulldown of His-SUMO-PPM1H on Cobalt-resin]
Resuspend the Cobalt-NTA-resin using a P1000 with such a modified blue tip and combine the resin into a  centrifuge vial.
15 mL
?. [Preparation of cell lysate and pulldown of His-SUMO-PPM1H on Cobalt-resin]
Wash out any remaining agarose from the large vials with  of wash buffer and pool with the first batch to maximise recovery.
1 mL
?. [Preparation of cell lysate and pulldown of His-SUMO-PPM1H on Cobalt-resin]
Fill the  vial to the top with wash buffer, mix well and sediment resin by centrifugation at  for  using an Eppendorf 5810 R centrifuge.
15 mL
Centrifuge: 1000 34
?. [Preparation of cell lysate and pulldown of His-SUMO-PPM1H on Cobalt-resin]
Remove the wash buffer with a thin vacuum line and replace with fresh wash buffer.  Repeat this step 5 times in total to thoroughly wash the resin.
?. [Preparation of cell lysate and pulldown of His-SUMO-PPM1H on Cobalt-resin]
Remove all wash buffer without disturbing the agarose bed.
?. [Preparation of cell lysate and pulldown of His-SUMO-PPM1H on Cobalt-resin]
Resuspend the resin into  wash buffer, transfer into a BIORAD Polyprep column and let the buffer run out.
3 mL
?. [Preparation of cell lysate and pulldown of His-SUMO-PPM1H on Cobalt-resin]
Add  of elution buffer and let the void of the bed run out.
1 mL
?. [Preparation of cell lysate and pulldown of His-SUMO-PPM1H on Cobalt-resin]
Keep the sample volume small to avoid unnecessary protein concentration steps before the subsequent size exclusion.
?. [Preparation of cell lysate and pulldown of His-SUMO-PPM1H on Cobalt-resin]
Then elute the protein with  elution buffer into a  centrifuge vial.
5 mL
15 mL
This should provide   of protein solution of  -   (  -   total protein).
?. [Removal of the His-SUMO-tag with His-SENP1 (415-644)]
For each  of eluted PPM1H, mix with  of His-SENP1.
For certain applications it may be desirable to remove the N-terminal 6His-SUMO-tag. This can be achieved by incubation of the eluted PPM1H with His-SENP1 (415-644).
10 mg
1 mg
?. [Removal of the His-SUMO-tag with His-SENP1 (415-644)]
Transfer the proteins into a Snake Skin dialysis tube and dialyse  against  of dialysis buffer.
5 L
Dialysis removes 95% of the imidazole and also concentrates the PPM1H, due to the higher glycerol concentration in the dialysis buffer.
?. [Removal of the His-SUMO-tag with His-SENP1 (415-644)]
Recover the protein the next day and remove the 6-His-SUMO-tag and the His-SENP1 by letting the proteins drip through a  bed of Cobalt-NTA-resin, equilibrate in dialysis buffer.
2 mL
?. [Removal of the His-SUMO-tag with His-SENP1 (415-644)]
Wash the bed with  of dialysis buffer and pool this wash with the flow through to maximise recovery.
2 mL
?. [Further purification by Size Exclusion Chromatography]
In order to further improve the purity of the PPM1H and to separate the active dimer from less active tetramer, employ SEC using a SD200 column.
?. [Further purification by Size Exclusion Chromatography]
Equilibrate a Superdex 200 XK 16/60 format in 3 column volumes of SEC buffer at a flowrate of  using an Äkta Purifier or an Äkta Pure.
1.2
?. [Further purification by Size Exclusion Chromatography]
Apply  aliquots of the PPM1H sample (typical concentration of -) to the column at a flowrate of  and collect   fractions between  (void) to .
1.5 mL
1.2
1.2 mL
44 mL
110 mL
?. [Further purification by Size Exclusion Chromatography]
Elute the tetrameric PPM1H as a shoulder between  and .
60 mL
75 mL
?. [Further purification by Size Exclusion Chromatography]
The dimeric PPM1H elutes as a peak between  and  ( apex at  ).
75 mL
87 mL
81 mL
We do not observe a monomer of PPM1H.
?. [Further purification by Size Exclusion Chromatography]
Pool and concentrate the fractions from  to  to obtain the dimeric PPM1H.
77 mL
87 mL
A typical elution chromatogram for PPM1H is shown on Figure 1a and for PPM1H (33-end).
?. [Quality control]
Separate  of the protein on a Bis-Tris 4% - 12% SDS-polyacrylamide gel and stain with Instant Blue Figure 2.
3 µg