TITLE: Expression	and	purification	of	Rab10 (1-181)	stoichiometrically	 phosphorylated	at	Thr73 (the	LRRK2	site)
AUTHORS: Axel Knebel , Kerryn Berndsen, Pawel Lis, Dario Alessi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Rab10 (Uniprot: P61026), a membrane associated small GTPase, likely involved in vesicle trafficking, has been identified as one of the 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 studies of Rab10 phosphorylation and dephosphorylation, it is necessary to produce milligram amounts of pure, stoichiometrically phosphorylated Rab10 protein. The full-length sequence of Rab10 is very prone to aggregation and precipitation when expressed in bacteria, resulting in extremely low yields of protein, even when solubilising tags are employed for the expression of fusion proteins. A shorter fragment, spanning residues 1-181 is much more useful for bacterial expression. Here we describe in detail the method we use to produce milligram quantities of stoichiometrically Thr73 phosphorylated Rab10[1-181]. We employ the MST3 kinase to phosphorylate recombinant Rab10 at Thr73, because MST3 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 Rab10 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 overnight 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 Rab10 expression by supplementing the medium with  IPTG, e.g. with  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 .
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 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.
25 mL
?. [Collection of cells and preparation of lysate]
If any of the sediments has not well resuspended, pipette up and down close to the bottom of the pots.
?. [Collection of cells and preparation of lysate]
Supplement NaCl to  final concentration and add glycerol to 10% final concentration and mix well.
?. [Collection of cells and preparation of lysate]
Aliquot  into  centrifuge vials and freeze them in liquid nitrogen for .
45 mL
50 mL
?. [Collection of cells and preparation of lysate]
Store at  for up to .
-20 °C
The freezing and subsequent thawing step breaks up the cells and improves yield.
?. [Preparation of cell lysate and pulldown of His-Rab10 on Ni-agarose]
Slowly thaw the vials with the cell suspension in cold water.
?. [Preparation of cell lysate and pulldown of His-Rab10 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-Rab10 on Ni-agarose]
Settings: 6 – 8 pulses of  with  pauses. Set the amplitude to 50%.
?. [Preparation of cell lysate and pulldown of His-Rab10 on Ni-agarose]
Transfer the sonicated suspension into  Beckman polypropylene 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.
50 mL
Centrifuge: 40000 34
4 °C
?. [Preparation of cell lysate and pulldown of His-Rab10 on Ni-agarose]
Recover the supernatants by carefully decanting them into a  Corning PP conical centrifuge tube.
500 mL
?. [Preparation of cell lysate and pulldown of His-Rab10 on Ni-agarose]
During the centrifugation step equilibrate  Ni-agarose  of a 50% slurry, sufficient for a  expression) by washing it three times with Milli Q water and once with cell collection buffer.
3 mL
6 mL
12 L
?. [Preparation of cell lysate and pulldown of His-Rab10 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
?. [Preparation of cell lysate and pulldown of His-Rab10 on Ni-agarose]
Avoid excessive agitation and especially formation of foam.
?. [Preparation of cell lysate and pulldown of His-Rab10 on Ni-agarose]
In the meanwhile, prepare and chill the Ni-wash buffer.
?. [Preparation of cell lysate and pulldown of His-Rab10 on Ni-agarose]
Carefully sediment the Ni-agarose by centrifugation using a Beckman J6 with a 4.2 rotor and suitable adaptors.
?. [Preparation of cell lysate and pulldown of His-Rab10 on Ni-agarose]
Centrifuge at  for  at .
Centrifuge: 1000 33
4 °C
?. [Preparation of cell lysate and pulldown of His-Rab10 on Ni-agarose]
Remove the lid and carefully remove 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-Rab10 on Ni-agarose]
Add  of Ni-wash buffer.
6 mL
?. [Preparation of cell lysate and pulldown of His-Rab10 on Ni-agarose]
Prepare a  pipette tip by removing  –  from the pointed end using scissors.
1000 µl
5
7
This allows it to be used to facilely resuspend the agarose.
?. [Preparation of cell lysate and pulldown of His-Rab10 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-Rab10 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-Rab10 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-Rab10 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-Rab10 on Ni-agarose]
Remove all Ni-wash buffer without disturbing the agarose bed and add  of Ni-wash buffer.
1 mL
?. [Preparation of cell lysate and pulldown of His-Rab10 on Ni-agarose]
Add 100U =  Thrombin solution (1000 Units per ml) to the Ni-agarose and mix carefully but well.
100 µl
?. [Preparation of cell lysate and pulldown of His-Rab10 on Ni-agarose]
Incubate the Ni-agarose with Thrombin for  at ambient temperature (  –  ) and mix occasionally.
20 °C
24 °C
?. [Preparation of cell lysate and pulldown of His-Rab10 on Ni-agarose]
Transfer the Ni-agarose into a Biorad  Polyprep column and let the digested protein drip into a fresh  vial.
5 mL
15 mL
?. [Preparation of cell lysate and pulldown of His-Rab10 on Ni-agarose]
Wash out the original  vial with  Ni-wash buffer and pool with the Ni-agarose in the Polyprep column. This improves recovery.
15 mL
2 mL
?. [Preparation of cell lysate and pulldown of His-Rab10 on Ni-agarose]
Finally, after the Ni-agarose has settled down add another  of Ni-wash buffer to recover any remaining digested protein.
2 mL
At this stage there should be   –   of a protein solution at   -  .
?. [Isolation of monomeric Rab10 (1-181) by Size Exclusion Chromatography]
Equilibrate a Superdex 75 HiPrep (XK 16/60) column in SEC buffer I.
The recovered, untagged Rab10 is a mix of aggregated Rab10 (MW > 100 kDa), Thrombin (native Mw 37 kDa) and monomeric Rab10 1-181 (Mw 22.9 kDa).
?. [Isolation of monomeric Rab10 (1-181) by Size Exclusion Chromatography]
Apply the digested Rab10 sample either in 2 x  or as 1 x  to the column and develop the column at a flowrate of .
3 mL
6 mL
1.2
We normally observe a substantial amount of protein eluting at the void volume and thereafter.
?. [Isolation of monomeric Rab10 (1-181) by Size Exclusion Chromatography]
However, seperate the monomeric Rab10 from these aggregates and contaminants and elutes at around  (Figure 1). Collect the fractions of this peak.
84 mL
We normally obtain   –   of monomeric Rab10 from such preparation from   of bacterial culture.
?. [Isolation of monomeric Rab10 (1-181) by Size Exclusion Chromatography]
Pool and concentrate the protein using Amicon Ultra 3000 Da MWCO filters.
The protein tolerates concentration to   and more. It can be frozen in liquid nitrogen and stored at  .
?. [Phosphorylation]
Mix  ( of Rab10 protein at ) in SEC buffer I with  of His-MST3 protein ( ),  Tris  ( f.c.),  MgCl2 (  f.c.), and  ATP ( f.c.) and incubate at  for  – .
In order to produce   of phosphorylated Rab10, it is necessary to phosphorylate   -  of purified Rab10 protein and then repurify the phosphorylated species. Hence the Rab10 protein from   of bacterial culture is required and must be prepared first. We combine the Rab10 protein from three   preparations for this purpose.
10 mg
4 mL
2.5 mg
4 mg
0.25 mL
55 µl
55 µl
28 °C
?. [Phosphorylation]
After the phosphorylation reaction is completed, remove the kinase by depletion over a  Ni-agarose bed.
Although MST3 is not a very potent Rab10 kinase, 90% of the Rab protein does become phosphorylated in these conditions.
1 mL
Some losses occur, but   of phosphorylated Rab10 can be recovered.
?. [Repurification and buffer exchange by Size Exclusion Chromatography]
Apply the protein to a Superdex 75 column, equilibrate this time in SEC buffer II.
This step removes the ATP and ADP, any remaining MST3 and replaces the buffer system. There are two important changes compared to SEC buffer I: firstly the buffer system is MES at   and not Tris at  . This is to protonise the Rab10 protein in preparation for the next cation exchange step and Tris is not a suitable buffer system for cation exchange chromatography. Secondly, SEC buffer II does not contain L-arginine, which would interfere with the subsequent cation-exchange step. Figure 2 shows that the pRab10 protein elutes at  , exactly where the Rab10 would elute.
?. [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]
Therefore, dilute the monomeric Rab10 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]
Equilibrate the Source 15 S HR10/10 with the IEX- buffers using an Äkta Pure or Purifier.
?. [Chromatography on a Source 15 S HR10/10 column to separate phospho species]
Dilute the pRab10 protein isolated from the gel filtration step tenfold into the IEX-Low Salt buffer.
?. [Chromatography on a Source 15 S HR10/10 column to separate phospho species]
Apply aliquots equivalent to  to the S-column at a flowrate of .
3 mg
2
Two column runs may be necessary.
?. [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 3. The dominant peak eluting at 15 mS/cm (here  ) represents single phosphorylated Rab10.
?. [Chromatography on a Source 15 S HR10/10 column to separate phospho species]
Pool the fractions containing pRab10 protein.
Protein yield should exceed  .
?. [Chromatography on a Source 15 S HR10/10 column to separate phospho species]
Supplement GTPgS to  or GDP to  as required.
?. [Chromatography on a Source 15 S HR10/10 column to separate phospho species]
Aliquot into convenient batches, freeze in liquid nitrogen and store at .
-70 °C
?. [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 a  of unphosphorylated Rab10 (1-181) and  of unphosphorylated Rab10 (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 4b).
3 µg
3 µg
The migration of the phosphorylated and non-phosphorylated pRab10 proteins can clearly be distinguished.