TITLE: Expression and purification of Tribolium castaneum orthologue of PINK1
AUTHORS: Olawale G. Raimi, Miratul M. K. Muqit

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Mutations in PINK1 (protein kinase) and Parkin (ubiquitin E3 ligase) have been linked to familial early-onset Parkinson’s disease. PINK1 phosphorylates ubiquitin and the N-terminal ubiquitin-like domain (Ubl) of Parkin at a conserved Serine65 residue in both proteins to initiate mitophagy. Direct measurement of human PINK1 kinase activity has been challenging due to poor expression and low activity in vitro. The discovery of catalytically active insect PINK1 orthologues including Tribolium castaneum PINK1 (TcPINK1) and Pediculus humanus corporis PINK1 (PhcPINK1) has enabled elaboration of robust assays for measurement of PINK1 kinase activity (Woodroof et al., 2011). These assays can be employed for the study of human pathogenic PINK1 mutations and in vitro structural and functional studies of PINK1 (Kumar et al., 2017). Herein, we describe in detail a protocol for expression and purification of TcPINK1 for crystallisation as well as for in vitro characterisation. </div></div>

[STEPS]
?. [Transformation of competent bacteria]
Mix  of pET 6His sumo codon optimised TcPINK1 150-end ΔI261-L270 S205E E527A K528A plasmid () with of the competent BL21(DE3) cells.
1 µl
50 µl
?. [Transformation of competent bacteria]
Incubate  for .
on ice
?. [Transformation of competent bacteria]
Heat shock the cells in a water bath at  for .
42 °C
?. [Transformation of competent bacteria]
Transfer the vial back onto ice and add  SOC and mix gently.
200 µl
?. [Transformation of competent bacteria]
Incubate for at  for recovery.
37 °C
?. [Transformation of competent bacteria]
Plate all of the cells onto a LB-agar plate supplemented with  ampicillin.
?. [Transformation of competent bacteria]
Leave the plate overnight in a  incubator.
37 °C
?. [Making overnight culture]
Pick a single colony using a sterile pipette tip from the plate and drop the tip into  LB medium, supplemented with  ampicillin.
100 mL
?. [Making overnight culture]
Incubate at  with  -  rotational shaking .
37 °C
Centrifuge: 180 33
Centrifuge: 200 33
The culture medium should become totally dense or turbid the following morning.
?. [Protein expression]
Decant 6 x 1L TB medium into 6 x 2L autoclaved conical flasks.
?. [Protein expression]
To each flask add  of ampicillin per litre.
50 µg
Prepare the stock antibiotics as 1000- fold to ensure consistent supplementation.
?. [Protein expression]
After  check the OD600 by taking  of randomly selected flasks and a fresh TB medium as blank using a WPA cell densitometer or a spectrometer.
1 mL
?. [Protein expression]
When the temperature drops to , induce protein expression by adding  of IPTG (From 1 M stock) and allow the cells to continue to grow for a further 16 - 20 h (usually ).
16 °C
100
?. [Harvesting of cells]
The following morning, decant the content of the 2 L conical flasks into 1 L Beckman centrifuge pots.
?. [Harvesting of cells]
At the end of the run, carefully decant the supernatant back into the flasks.
The flasks can then be autoclaved and cleaned.
?. [Harvesting of cells]
The cell pellets (usually between – volume) are then resuspended using the lysis buffer.
3 mL
5 mL
?. [Harvesting of cells]
To do this,  of cold lysis buffer is added to the pellet in each of the pot supplemented with  of a  Leupeptin solution, of a 0.5 M Pefabloc solution and  of  DNase
10 mL
30 µl
60 µl
100 µl
?. [Harvesting of cells]
Transfer the  pots to the Infors incubator and the temperature still at  and the rotation lowered to . Leave the pots to rotate for , after which the cell pellet should have completely resuspended.
1 L
16 °C
Centrifuge: 120 33
If there are still some pellet left, pipette up and down close to the bottom of the pots.
?. [Harvesting of cells]
Transfer the resuspended cells into  falcon tubes with aliquots and flash freeze in liquid nitrogen (this helps to also break up the cells) and stored in  for short time storage or in  for longer storage.
50 mL
30 mL
-20 °C
-80 °C
?. [Preparation of cell lysate and pulldown]
When you are ready for the purification, slowly thaw the cells suspension on or in cold water.
Best to prepare all the buffers a day before and keep in the cold room
on ice
Make sure the cells are   always or best work in the cold room
?. [Preparation of cell lysate and pulldown]
Once the cells are resuspended move to lyse the cells using a sonicator or cell disruptor. Sonicate , using a probe sonicator (Settings: 6 – 8 pulses of  with  pauses. Set the amplitude to 45%.
on ice
Please note the lysate will warm up slightly, but make sure it does not warm up over
?. [Preparation of cell lysate and pulldown]
Transfer the sonicated suspension into  Beckman centrifuge tubes and collect the cell debris by centrifugation at  for  at  using a 25.50 rotor in a Beckman Avanti centrifuge.
40 mL
4 °C
Centrifuge: 20.000 34
?. [Preparation of cell lysate and pulldown]
Load  Ni-agarose slurry on a  gravity column and wash three times with Milli Q water and once with the binding buffer.
5 mL
60 mL
5 ml of a 50% slurry, sufficient for a 6 L expression
?. [Preparation of cell lysate and pulldown]
Add the Ni-agarose slurry to the cell lysate in an appropriate size Duran bottle and incubate the mix on a roller shaker for  in the cold room.
Do not filter the lysate
?. [Preparation of cell lysate and pulldown]
After the incubation period, collect the beads by passing the mixture through a gravity flow chromatography column, wash the beads thoroughly using the washing buffer.
60 mL
?. [Preparation of cell lysate and pulldown]
Once clean elute the protein using the elution buffer.
Check that all the protein has been eluted by checking with Coomassie
?. [Preparation of cell lysate and pulldown]
Cleave PINK1 from His-sumo by dialysing the eluted protein against the dialysis buffer using the 3.5 kDa dialysis bag in the presence of His-Snep1 protease in the cold room.
1 mg of His-SENP1 per 25 mg of PINK1
The following day perform negative pull down to get rid of snep1 and sumo.
?. [Negative pull down]
To remove His-sumo and His-senp1 from the cleaved protein, perform negative pull down.
?. [Negative pull down]
To do this, still in the cold room, pack a PolyPrep chromatography column with nickel beads, equilibrate with binding buffer and gently by pipetting pass the dialysed protein mixture through the packed Ni-beads.
5 mL
2 mL
This traps the Histidine tagged proteins and your cleaved PINK1 protein passes through
?. [Negative pull down]
Collect the flow through into a new falcon tube.
?. [Concentration of the protein]
To concentrate the protein, consider using PEG20,000. To do this, the protein is dialysed against the PEG dialysis buffer. This takes between  to  depending on the starting volume
This is the tricky bit as the protein tends to aggregate/oligomerises and this make concentrating the protein using the vivaspin concentrator a bit difficult (increased agitation of the protein due to long concentration time).
Immerse the dialysis bag with protein in some peg2000 powder will equally do the magic in reducing the volume
The idea is that you want to reduce the volume drastically before moving to vivaspin concentrator to reduce the level of agitation on the protein.
?. [Size Exclusion Chromatography]
Equilibrate the column with 2-column volume of the column size with the SEC buffer.
To obtain a crystallization grade protein, size exclusion chromatography is always the last purification step. This can be done on any of the Akta systems at   on a superdex75 pg (26/600) or superdex200 pg 16/600 column
Before loading the sample clean the Akta sample loop with 2CV of the loop size first with water followed by the SEC buffer.
?. [Size Exclusion Chromatography]
Inject the sample and run the program (usually 1 ml/min flowrate and fractions).
3 mL
Using this program on superdex 75 column gives a chromatogram shown in Figure 1a.
?. [Purity check]
Run of fractions within the peak (Figure 1a) on a 12% Tris Glycine SDS-polyacrylamide gel at the end of the run and stain with small amount Instant Blue.
5 µl
?. [Purity check]
After detaining a single band corresponding to ~ 48 kDa should be visible as shown in Figure 1b.
?. [Quantification and storage]
Pull together the cleanest fractions within the peak and concentrate using the vivaspin 30 kDa cut off to at least or less this might take between  -  depending on your initial volume.
Here it is expected that you have less volume, however if your volume is large you might consider using the PEG method to reduce the volume before finishing off with vivaspin concentrator.
?. [Quantification and storage]
Make aliquots and flash freeze in liquid nitrogen and stored in.
-80 °C
?. [Protein expression]
Incubate at , using an Infors Shaker-Incubator set at .
37 °C
Centrifuge: 180 33
?. [Protein expression]
Then add  of the  culture into each flask.
10 mL
?. [Protein expression]
When the OD600 has reached 0.6 - 0.8, lower the temperature of the incubator to  and continue the incubation for at least another .
16 °C
?. [Harvesting of cells]
Close the pots tightly and sediment the cells at  by centrifugation for  at  using a Beckman J6 centrifuge with the 6 x 1 L rotor.
4 °C
Centrifuge: 4000 33
?. [Preparation of cell lysate and pulldown]
Check that the beads are clean by adding of the flow through to of Coomassie.
20 µl
100 µl