TITLE: Expression	and	purification	of	Rab12 (1-244)	stoichiometrically	 phosphorylated	at	Ser106 (the	LRRK2	site)
AUTHORS: Axel Knebel , Pawel Lis, Dario Alessi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A subset of small GTPases of the Rab family including Rab12 (Uniprot: Q6IQ22) have been identified as substrates of the Leucine Rich Repeat Kinase 2 (LRRK2; Uniprot Q5S007) (Steger et al., 2016). In order to perform detailed study of Rab phosphorylation and dephosphorylation, as well as produce phosphorylation site-specific antibodies, and to carry out drug discovery screens, it is necessary to produce hundreds of milligrams of pure, stoichiometrically phosphorylated Rab12 protein. The full-length sequence of Rab12 (1-244) is suitable for large scale expression, when expressed as a 6His-SUMO fusion protein. Here we describe in detail the method we use to produce milligram quantities of stoichiometrically Ser106 phosphorylated Rab12 with the 6His-SUMO tag removed employing the SENP1 protease. We employ the MST3 kinase to phosphorylate Rab12 at Ser106, as this kinase is much easier and less expensive to produce or purchase than LRRK2 (Berndsen et al., 2019, Vieweg et al. 2020).</div></div>

[STEPS]
?. [Transformation of plasmid into competent bacteria]
Mix  of pET15b-6HisSUMO-Rab12 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  pipette tip, remove one of the colonies from the plate and drop the tip into  LB broth medium, supplemented with  carbenicillin.
200 µl
300 mL
?. [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 6 x  LB broth medium into 6 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 around  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 .
15 °C
Centrifuge: 200 33
?. [Set up cells and induce expression]
When the flasks have cooled down to  or lower, induce His-SUMO-Rab12 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 .
15 °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 screwcap lids and sediment the cells by centrifugation for  at  at  using a Beckman J6 centrifuge with the 6 x 1 L rotor (4.2).
2 L
1 L
4 °C
Centrifuge: 4200 33
?. [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 6 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
?. [Collection of cells and preparation of lysate]
For Rab proteins, 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, aliquot  samples 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  for up to . The freezing and subsequent thawing step breaks up the cells and improves yield.
-20 °C
?. [Preparation of cell lysate and pulldown of His-SUMO-Rab12 on Ni-agarose]
Slowly thaw the vials with the cell suspension in cold water.
We have verified that it is OK to leave the tubes to thaw samples by leaving these at    .
?. [Preparation of cell lysate and pulldown of His-SUMO-Rab12 on Ni-agarose]
After thawing chill suspension  and then sonicate, using a probe sonicator (Cell disruptor).
on ice
?. [Preparation of cell lysate and pulldown of His-SUMO-Rab12 on Ni-agarose]
Settings: 6 – 8 pulses of  with  pauses. Set the amplitude to 50%.
?. [Preparation of cell lysate and pulldown of His-SUMO-Rab12 on Ni-agarose]
The lysate will warm up slightly, but make sure it does not warm up over  and keep samples cool by swirling .
18 °C
on ice
?. [Preparation of cell lysate and pulldown of His-SUMO-Rab12 on Ni-agarose]
Transfer the sonicated suspension into  Beckman centrifuge vials and sediment the insoluble material by centrifugation for  at  an  using a 25.50 or a 30.50 rotor in a Beckman Avanti centrifuge.
40 mL
Centrifuge: 40000 34
4 °C
?. [Preparation of cell lysate and pulldown of His-SUMO-Rab12 on Ni-agarose]
Recover the supernatant by carefully decanting it into a  Corning PP conical centrifuge tube.
500 mL
?. [Preparation of cell lysate and pulldown of His-SUMO-Rab12 on Ni-agarose]
During the centrifugation step, equilibrate  Ni-agarose ( of a 50% slurry, sufficient for a 6 L expression) by washing it three times with Milli Q water and once with cell collection buffer.
3 mL
6 mL
?. [Preparation of cell lysate and pulldown of His-SUMO-Rab12 on Ni-agarose]
Add a 50% slurry of the washed Ni-agarose in collection buffer to the lysate and incubate the mix on a Roller Mixer for  in a cold room set at .
4 °C
Avoid excessive agitation and especially formation of foam.
?. [Preparation of cell lysate and pulldown of His-SUMO-Rab12 on Ni-agarose]
In the meanwhile, prepare Ni-wash buffer.
?. [Preparation of cell lysate and pulldown of His-SUMO-Rab12 on Ni-agarose]
Carefully sediment the Ni-agarose by centrifugation using a Beckman J6 with a 4.2 rotor and suitable adaptors. Centrifuge at  for  at  .
Centrifuge: 1000 33
4 °C
Avoid higher centrifugation speeds as the   PP conical centrifuge vessels are structurally weak.
?. [Preparation of cell lysate and pulldown of His-SUMO-Rab12 on Ni-agarose]
Remove the lid and carefully decant the supernatant containing the depleted lysate using a  pipette, being careful not to disturb the Ni-agarose.
25 mL
?. [Preparation of cell lysate and pulldown of His-SUMO-Rab12 on Ni-agarose]
Add  of Ni-wash buffer.
6 mL
?. [Preparation of cell lysate and pulldown of His-SUMO-Rab12 on Ni-agarose]
Prepare a  pipette tip by removing  –  from the pointed end using scissors. This allows it to be used to facilely resuspend the agarose.
1000 µl
5
7
?. [Preparation of cell lysate and pulldown of His-SUMO-Rab12 on Ni-agarose]
Resuspend the Ni-agarose using a P1000 with such a modified blue tip and aliquot the Ni-agarose into a  centrifuge vial.
15 mL
?. [Preparation of cell lysate and pulldown of His-SUMO-Rab12 on Ni-agarose]
Wash out any remaining agarose from the large vial with  of Ni-wash buffer and pool with the first batch to maximise recovery.
1 mL
?. [Preparation of cell lysate and pulldown of His-SUMO-Rab12 on Ni-agarose]
Fill the  vial to the top with Ni-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-Rab12 on Ni-agarose]
Remove the Ni-wash buffer with a thin vacuum line and replace with fresh Ni-wash buffer. Repeat this step 5 times in total to thoroughly wash the resin.
?. [Preparation of cell lysate and pulldown of His-SUMO-Rab12 on Ni-agarose]
Remove all Ni-wash buffer without disturbing the agarose bed.
?. [Preparation of cell lysate and pulldown of His-SUMO-Rab12 on Ni-agarose]
Resuspend the Ni-agarose into  wash buffer, transfer into a BIORAD Econopac column and let the buffer run out.
5 mL
?. [Preparation of cell lysate and pulldown of His-SUMO-Rab12 on Ni-agarose]
Add  of Ni-elution buffer and let the void of the bed run out.
1 mL
?. [Preparation of cell lysate and pulldown of His-SUMO-Rab12 on Ni-agarose]
Then elute the protein with  Ni-elution buffer into a  centrifuge vial.
6 mL
15 mL
This should provide   of protein solution of   -   (  –   total protein).
?. [Polishing and buffer exchange by SEC]
In order to improve protein purity and to remove the imidazole prepare a Superdex 75 XK16/60 column by equilibration into SEC buffer .
?. [Polishing and buffer exchange by SEC]
Apply  of the His-SUMO-Rab12 and perform chromatography on that column. The His-SUMO-RAB12 elutes at  (Figure 1). Pool the peak fractions.
5 mL
70 mL
?. [Removal of His-SUMO-tag and phosphorylation by MST3]
Proceed by taking two aliquots, one to be left unphosphorylated and another to be phosphorylated.
?. [Removal of His-SUMO-tag and phosphorylation by MST3]
To remove the tag, add His-SENP1 protease to the His-SUMO-Rab12 aliquots at a ratio of  of protease per  of substrate.
1 mg
5 mg
?. [Removal of His-SUMO-tag and phosphorylation by MST3]
To the aliquot to be phosphorylated also add  of His-MST3 kinase and make the solution   MgCl2 and  ATP ().
2 mg
?. [Removal of His-SUMO-tag and phosphorylation by MST3]
Incubate at .
28 °C
?. [Removal of His-SUMO-tag and phosphorylation by MST3]
Prepare two Biorad  Polyprep filters with  Ni-agarose beds, equilibrate in wash buffer.
5 mL
1.5 mL
?. [Removal of His-SUMO-tag and phosphorylation by MST3]
Individually pass the proteins through these filters, in order to remove the His-tagged enzymes and the cleaved His-SUMO-tag.
?. [Removal of His-SUMO-tag and phosphorylation by MST3]
For further purification and removal of ATP, equilibrate a Superdex 75 HR10/30 column into SEC buffer
?. [Removal of His-SUMO-tag and phosphorylation by MST3]
Apply the Rab12 samples and collect fractions.
The cleaved Rab12 protein elutes at  -  peaking at   (Figure 2).
?. [Chromatography on a Source 15 S HR10/10 column to separate phospho  species]
In order to separate the phospho species from each other and from the remaining unphosphorylated protein, employ cation exchange chromatography using a Source 15 S column.
?. [Chromatography on a Source 15 S HR10/10 column to separate phospho  species]
To this end, pack an empty HR10/10 or GL 10/100 column with  Source 15 S resin and use vacuum suction to obtain a homogenous well packed resin bed.
10 mL
Ion exchange chromatography is a concentrating method, hence the load volume is not critical.
?. [Chromatography on a Source 15 S HR10/10 column to separate phospho  species]
Dilute the aliquots of the Rab12 samples into the Low Salt buffer to reduce the ionic strength sufficiently for the protein to bind.
?. [Chromatography on a Source 15 S HR10/10 column to separate phospho  species]
Equilibrate the Source 15 S HR10/10 with the IEX- buffers using an Akta Pure or Purifier.
?. [Chromatography on a Source 15 S HR10/10 column to separate phospho  species]
Dilute the Rab12 protein isolated from the gel filtration step tenfold into the IEX-LowSalt buffer.
?. [Chromatography on a Source 15 S HR10/10 column to separate phospho  species]
Apply aliquots equivalent up to  to the S-column at a flowrate of .
6 mg
2
?. [Chromatography on a Source 15 S HR10/10 column to separate phospho  species]
Develop the column at the same flow rate with a shallow  gradient to 30% IEX-High Salt buffer collecting  fractions.
100 mL
1 mL
pRab12 eluted as a major peak with a conductivity of 15 mS/cm, equivalent to   NaCl ( Figure 3). Our QC analysis indicates that the major peak is mono phosphorylated Rab12. The minor peak to the left is double phosphorylated Rab12.
?. [Quality control]
Separate  of the protein on a 4% - 20% Tris Glycine SDS polyacrylamid gel and stain with Instant Blue (Figure 4a).
3 µg
The protein should be >95% homogeneous.
?. [Quality control]
Separate  of pRab12 and  of phosphorylated Rab12, which had been treated with alkalyne phosphatase on a 12% Phos-tag SDS-Polyacrylamid gel that separates phosphorylated and non-phosphorylated Rab proteins, run as described previously (Ito et al. 2016) (Figure 4b).
3 µg
3 µg
The migration of the phosphorylated and non-phosphorylated pRab12 proteins can clearly be distinguished.
?. [Quality control]
Do Immunoblot analysis of pRab12 and dephosphorylated Rab12 using Rab12 pSer106 and total antibodies (Figure 5). This will reveal that the pRab12 protein is specifically phosphorylated at Ser106.Figure 5. Indicated amounts of pRab12 (P) and dephosphorylated Rab12 (NP) were analysed using Phostag SDS-PAGE (Coomassie stained as well as immunoblotted with Rab12 total/pS106 antibodies), and SDS-PAGE (immunoblotted with Rab12 total/pS106 antibodies).