TITLE: Expression	and	purification	of	Rab8A	(1-181)	stoichiometrically	 phosphorylated	at	pThr72 (the	LRRK2	site)
AUTHORS: Axel Knebel, Kerryn Berndsen, Pawel Lis, Paul Davies, Dario Alessi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A subset of small GTPases of the Rab family including Rab8A (Uniprot: P61006) have been identified as substrates of the Leucine Rich Repeat Kinase 2 (LRRK2; Uniprot Q5S007)) and the Protein Phosphatase PPM1H (Uniprot Q9ULR3) (Steger et al., 2016; Berndsen et al., 2019). 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 Rab8A protein. The full-length sequence of Rab8A is prone to aggregation and precipitation when expressed in bacteria. Therefore, a shorter fragment, spanning residues 1-181 is more useful for large scale expression. Here we describe in detail the method we use to produce milligram quantities of stoichiometrically Thr72 phosphorylated Rab8A[1-181]. We employ the MST3 kinase to phosphorylate Rab8A at Thr72, 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 pET28a 6HIS Thrombin Rab8a 1-181 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 kanamycin.
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 kanamycin.
200 µl
300 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 12 x  LB broth medium into 12 x  conical flasks.
1 L
2 L
?. [Set up cells and induce expression]
Supplement each flask/litre with of  Kanamycin Monosulphate.
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 600 nm with fresh LB medium, using a WPA cell densitometer or a spectrometer.
1 mL
?. [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 , all the while shaking at .
18 °C
Centrifuge: 200 33
?. [Set up cells and induce expression]
When the flasks have cooled down to  or lower, induce Rab8A expression by supplementing the medium with  IPTG. This is achieved by adding of a  IPTG stock solution to each litre.
20 °C
100 µl
?. [Set up cells and induce expression]
Leave the cells to express the protein for at .
18 °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  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 12 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 Rab8A and other Rab proteins is it 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
The vials can be stored at   for at least 4 weeks. The freezing and subsequent thawing step breaks up the cells and improves yield.
?. [Preparation of cell lysate and pulldown of His-Rab8A 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-Rab8A on Ni-agarose]
After thawing chill suspension on ice and then sonicate, using a probe sonicator (Cell disruptor). Settings: 6-8 pulses of  with 15 sec pauses. Set the amplitude to 50%.
?. [Preparation of cell lysate and pulldown of His-Rab8A on Ni-agarose]
The lysate will warm up slightly, but make sure it does not warm up over  and keep samples cool by swirling on ice.
18 °C
?. [Preparation of cell lysate and pulldown of His-Rab8A 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-Rab8A 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-Rab8A on Ni-agarose]
During the centrifugation step equilibrate  Ni-agarose ( of a 50% slurry, sufficient for a 12 L expression) by washing it three times with Milli Q water and once with cell collection buffer.
5 mL
10 mL
?. [Preparation of cell lysate and pulldown of His-Rab8A 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-Rab8A on Ni-agarose]
In the meanwhile, prepare Ni-wash buffer. Carefully sediment the Niagarose by centrifugation using a Beckman J6 with a 4.2 rotor and suitable adaptors.
?. [Preparation of cell lysate and pulldown of His-Rab8A on Ni-agarose]
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-Rab8A 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-Rab8A on Ni-agarose]
Add  of Ni-wash buffer.
6 mL
?. [Preparation of cell lysate and pulldown of His-Rab8A on Ni-agarose]
Prepare a  pipette tip by removing 5 mm-7 mm from the pointed end using scissors.
1000 µl
This allows it to be used to facilely resuspend the agarose.
?. [Preparation of cell lysate and pulldown of His-Rab8A 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-Rab8A 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-Rab8A 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-Rab8A on Ni-agarose]
Remove the Ni-wash buffer with a thin vacuum line and replace with fresh Ni-wash buffer.
?. [Preparation of cell lysate and pulldown of His-Rab8A on Ni-agarose]
Repeat this step 5 times in total to thoroughly wash the resin.
?. [Preparation of cell lysate and pulldown of His-Rab8A on Ni-agarose]
Remove all Ni-wash buffer without disturbing the agarose bed.
?. [Preparation of cell lysate and pulldown of His-Rab8A 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-Rab8A on Ni-agarose]
Add  of Ni-elution buffer and let the void of the bed run out. Then elute the protein with  Nielution buffer into a  centrifuge vial.
2 mL
10 mL
15 mL
This should provide   of protein solution of  -   (  –   total protein).
?. [Preparation of cell lysate and pulldown of His-Rab8A on Ni-agarose]
Add  Thrombin solution (1000 Units per ml) and dialyse against  of Rab8A dialysis buffer using Thermo-Fisher Snake Skin 3500 MWCO.
100 µl
5 L
?. [Preparation of cell lysate and pulldown of His-Rab8A on Ni-agarose]
The next day recover the protein into a  vial.
15 mL
The dialysis step is important, as high imidazole concentrations inhibit the subsequent kinase reaction. The higher glycerol concentration during dialysis will reduce the volume of the sample by up to 20%. We keep the sample volume small to avoid unnecessary protein concentration steps before the subsequent size exclusion.
?. [Phosphorylation]
Take  of Rab8A as an unphosphorylated control. Add  His-MST3 (2.3 ml 3.5mg/ml) to the Rab8A protein and mix well.
0.5 mg
8 mg
?. [Phosphorylation]
Add  of  Tris  to buffer to ATP solution.
0.5 mL
?. [Phosphorylation]
Add  MgCl2 and  ATP and mix well but gently.
120 µl
120 µl
?. [Phosphorylation]
Leave with an incubator at .
28 °C
?. [Phosphorylation]
Next day prepare a  Ni-agarose bed and capture the His-tagged MST3 by letting the sample run through the bed.
2 mL
?. [Further purification by Size Exclusion Chromatography]
Equilibrate a Superdex 75 XK 26/60 format in 3 column volumes of SEC buffer at a flowrate of  using an Akta Pure.
The untagged Rab8A is a mix of aggregated pRab8A, MST3 (Mw 51.8 kDa), Thrombin (native Mw 37 kDa) and monomeric pRab8A 1-181 (Mw 22.9 kDa). Size Exclusion Chromatography using a Superdex 75 XK 26/60 column offers a simple and powerful solution for isolation of monomeric pRab8A 1-181. The species are separated so well, that we have somewhat overloaded the column and apply a sample representing 4% of the column volume instead of the recommended 1%.
2.5
?. [Further purification by Size Exclusion Chromatography]
Apply the sample - (typical concentration of -) to the Gel filtration at a flowrate of  and collect  fractions between  to .
12 mL
15 mL
2.5
1.5 mL
80 mL
220 mL
?. [Further purification by Size Exclusion Chromatography]
Elute the monomeric pRab8A as a 36 ml peak from  to  with the apex at around .
170 mL
206 mL
184 mL
?. [Further purification by Size Exclusion Chromatography]
Pool the fractions containing the core of the pRab8A peak into one vial. A typical elution chromatogram is shown on Figure 1.
?. [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
?. [Chromatography on a Source 15 S HR10/10 column to separate phospho  species]
Ion exchange chromatography is a concentrating method, hence the load volume is not critical. Therefore, dilute aliquots of the monomeric Rab8A sample 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]
In contrast, dialysing highly concentrated Rab8A into the Low Salt Buffer leads to substantial protein aggregation and precipitation.
?. [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 pRab8A 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 to 5-6mg to the S-column at a flowrate of .
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
Generally, up to four peaks are resolved see Figure 2. Our QC analysis indicates that the peaks C and D eluting between 6 and 8 mS/cm both represent Thr72 monophosphorylated Rab8A.
?. [Quality control]
Separate  of the protein on a 4% - 20% Tris v with Instant Blue (Figure 3).
3 µg
The protein should be >95% homogeneous.
?. [Quality control]
Separate a  of pRab8A[1-181] and  of unphosphorylated Rab8A (1-181) 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 4).
3 µg
3 µg
The migration of the phosphorylated and non-phosphorylated pRab8A proteins can clearly be distinguished.
?. [Quality control]
Immunoblot analysis of pRab8A[1-181] and dephosphorylated Rab8A[1-181] undertaken as described previously (dx.doi.org/10.17504/protocols.io.bsgrnbv6) using Rab8A pThr72 and total antibodies (Figure 5). This will reveal that the pRab8A protein is specifically phosphorylated at Thr72.
?. [Quality control]
Incubate  pRab8A[1-181] ±   o PPM1H (can be ordered from MRCPPU Reagents and Services, DU62835) in the presence of  MgCl2 for  at  prior to running a Phostag gel.
Recombinant PPM1H phosphatase should quantitatively dephosphorylate pThr72 phosphorylated Rab8A.
3 µg
0.05 µg
28 Room temperature
?. [Quality control]
The pRab8A[1-181] can also be analysed by Mass Spectrometry after trypsin digestion. The tryptic peptide encompassing Thr72 should only be mainly present in the phosphorylated form and levels of the non-phosphorylated peptide should be very low to undetectable.