TITLE: Expression and purification of recombinant human Parkin and pSer65 Parkin
AUTHORS: Michael Stevens, Miratul M. K. Muqit

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Mutations in PARK2 encoding Parkin are causal for early-onset Parkinson’s disease. Parkin is a ubiquitin E3 ligase and is activated by the PINK1 kinase to stimulate ubiquitin-dependent removal of damaged mitochondria via autophagy (mitophagy). The ability to generate recombinant Parkin in its inactive unmodified conformation and activated phosphorylated conformation represent important tools for studying the its regulation by PINK1-dependent phosphorylation as well as understanding how disease mutations impact on its function. This protocol describes the expression and purification of full-length human untagged Parkin along with how to make the phosphorylated protein.</div><br/><div class = "text-block">In cells the removal of malfunctioning organelles is important for cellular survival. Damaged mitochondria are removed via mitophagy upon activation of the PTEN induced kinase 1 (PINK1) / Parkin pathway. In healthy mitochondria PINK1 is cleaved by PARL and released into the cytoplasm where it is degraded by the N-end rule. In damaged mitochondria depolarisation of the mitochondrial membrane stabilises PINK1, causing it to accumulate on the outer mitochondrial membrane. Stabilised PINK1 phosphorylates Ser65 on both ubiquitin and the ubiquitin like (Ubl) domain of Parkin, this activated Parkin. Active Parkin ubiquitinates outer mitochondrial membrane proteins, leading to ubiquitin chains containing pSer65 ubiquitin. The formation of these chains on the mitochondria identifies it for removal by mitophagy. Loss of activity in either Parkin or PINK1 have been observed in cases of early-onset Parkinson’s disease.</div><div class = "text-block">Parkin is a RING-in-between-RING (RBR) E3 ubiquitin ligase containing a ubiquitin like (UBL) domain, three RING domains (referred to as RING0, RING1, and RING2 domains), and an in between ring (IBR) domain. Unmodified Parkin exists in an autoinhibited conformation, and phosphorylation of Ser65 on the Ubl domain along with binding to pSer65 ubiquitin causes a significant conformational change that activates Parkin. Previous studies have shown that a small purification tag on the N-terminus of Parkin can lead to constitutive activation, making the production of untagged Parkin an important tool in the study of its regulation and ubiquitylation activity. We describe a protocol for the generation of untagged recombinant Parkin using a His-SUMO tagged fusion construct. In addition we describe the additional  steps to produce phospho-Parkin. The yield of Parkin produced varies between 2 and 6 mg, and this is 1/3 lower for pSer65 Parkin. For pSer65 Parkin the stoichiometry of phosphorylation can vary from 60 to 80 %.</div></div>

[STEPS]
?. [Transformation of plasmid into competent bacteria]
Mix   with  . Coli and incubate   for  .
[6His-SUMO-Parkin plasmid stock (~50 ng/\ul)]
[competent BL21 DE3 pLysS Codon Plus E]
on ice
?. [Transformation of plasmid into competent bacteria]
Heat shock cells by incubation in a heat block equilibrated at   for   and return to ice for  .
42 °C
?. [Transformation of plasmid into competent bacteria]
Add  , pre heated to  , to the cells and incubate at   for  .
[SOC media]
37 °C
37 °C
?. [Transformation of plasmid into competent bacteria]
Centrifuge the culture at   to pellet cells and remove  , leaving  .
Centrifuge: 1000 34
[media]
200 µl
?. [Transformation of plasmid into competent bacteria]
Suspend cells in the remaining media and spread on LB agar plates supplemented with  ., incubate plates at  [  ].
[carbenicillin]
37 °C
?. [Starter Culture]
Using a P200 pipette tip remove a colony from the LB agar plate and transfer to  +   in a 500 ml conical flask.
[LB]
[carbenicillin]
?. [Starter Culture]
Incubate the culture  , the culture should be opaque after incubation.
Centrifuge: 180 33, 960 min, 37 10
?. [Protein Expression]
Inoculate 12 x  +  +  each in 2 L conical flasks with  and incubate at  .
[LB]
[carbenicillin]
[ZnCl cultures]
[starter culture]
37 °C
?. [Protein Expression]
Set the incubator temperature to   once cultures reach an OD600 of 0.3 - 0.4, after ~3 hours.
15 °C
?. [Protein Expression]
When the incubator temperature is below   and the culture’s OD600 is 0.8- 0.95 add  to induce Parkin expression and incubate cultures  at  .
20 °C
[IPTG]
15 °C
?. [Protein Expression]
Incubators reduce temperatures at different rates, modify the OD600 at which the incubator temperature is lowered so cultures are at an OD600 of 0.8-0.95 when the incubator reaches   .
?. [Making Cell Lysate]
Pellet cells by centrifugation at  .
Centrifuge: 5020 34, 25 min, 4 10
?. [Making Cell Lysate]
Remove the media and invert the centrifuge bottles on a stack of paper towels, leave cell pellets to dry for  -  .
?. [Making Cell Lysate]
Suspend cell pellets in   containing   and   per 5 ml of cell pellet [the approximate pellet size from 1 L of cell culture].
[of ice-cold lysis buffer]
[leupeptin]
[AEBSF]
?. [Making Cell Lysate]
Transfer cell suspensions to 50 ml falcon tubes so that each tube contains  -  of solution.
30 mL
40 mL
?. [Making Cell Lysate]
Sonicate suspensions at 50 % amplitude  using  pulses with  breaks in between pulses for a total pulse time of  .
on ice
?. [Affinity Chromatography]
Pellet cell debris by centrifguation at   and transfer the supernatant to   in a 500 ml centrifuge pot.
Centrifuge: 35000 34, 30 min, 4 10
[Ni-NTA resin]
?. [Affinity Chromatography]
Incubate the solution with Ni-NTA resin at  -   on rollers for  to allow 6His-Sumo-Parkin to bind.
5 °C
7 °C
?. [Affinity Chromatography]
Centrifuge Ni-NTA resin at   to settle the resin, remove the supernatant, and suspend Ni-NTA resin in  .
Centrifuge: 500 34, 5 min, 4 10
[lysis buffer]
?. [Affinity Chromatography]
Transfer the suspension to 14 ml polypropylene tubes so to give 2 ml or less NiNTA resin per tube.
?. [Affinity Chromatography]
Settle Ni-NTA resin by centrifugation at   and aspirate the supernatant.
Centrifuge: 500 34, 1 min, 4 10
?. [Parkin S65 Phosphorylation]
Follow these additional steps if making pS65 Parkin, the protein preparation for pS65 Parkin takes an extra day over the wt Parkin protein preparation.
?. [Parkin S65 Phosphorylation]
Wash Ni-NTA resin six times by suspending in 6x the resin volume of kinase buffer, centrifuge at   to settle the Ni-NTA resin and aspirate the supernatant.
Centrifuge: 500 34, 1 min, 4 10
?. [Parkin S65 Phosphorylation]
Leave twice the Ni-NTA resin volume of kinase buffer after the final wash and add  to the Ni-NTA resin.
[GST pediculus humanus PINK1 (phPINK1)]
?. [Parkin S65 Phosphorylation]
Suspend the resin by inversion the leave at   for   without agitation, inverting occasionally, about every 30 minutes.
0 Room temperature
?. [Parkin S65 Phosphorylation]
Centrifuge the Ni-NTA suspension at  to settle and aspirate the solvent layer.
Centrifuge: 500 34, 1 min, 4 10
?. [Parkin S65 Phosphorylation]
Incubate Ni-NTA resin against 4 x the resin volume with kinase buffer containing 400 mM Imidazole.
?. [Parkin S65 Phosphorylation]
Transfer Ni-NTA resin to a gravity flow column and collect the flow through and wash the column in ¾ resin volume of cleavage buffer.
?. [Parkin S65 Phosphorylation]
Determine whether the protein has flowed through the column by comparing the colour change between  mixed with  and  mixed with  through at the point the buffer reaches the top of the resin bed.
[Bradford reagent]
[cleavage buffer]
[Bradford reagent]
[column flow]
?. [Parkin S65 Phosphorylation]
Add another ¾ resin volume of cleavage buffer if the flow through still has protein present.
?. [Parkin S65 Phosphorylation]
Check the protein concentration using a Bradford assay, add a 1:50 mass ratio of GST-PhcPINK1 to the elute and dialyse at  [  ] against 2 L kinase buffer.
0 Room temperature
?. [Parkin S65 Phosphorylation]
Incubate the kinase reaction with   at  -   for   and centrifuging at   and aspirate the supernatant.
[Ni-NTA resin]
5 °C
7 °C
Centrifuge: 500 34, 5 min, 4 10
?. [Affinity Chromatography]
Wash Ni-NTA resin by suspending in 6x the resin volume of lysis buffer, centrifuge at   to settle the Ni-NTA resin, and aspirate the supternatant.
Centrifuge: 500 33, 1 min, 4 10
?. [Affinity Chromatography]
Wash the resin four more times in lysis buffer then twice in cleavage buffer, keeping twice the resin volume of cleavage buffer in the tube after the final wash.
?. [Checking the Mass of Protein Bound to Resin]
Suspend the Ni-NTA resin in the leftover cleavage buffer and transfer  suspension to a 1.5 ml microcentrifuge tubes.
[Ni-NTA resin]
?. [Checking the Mass of Protein Bound to Resin]
Settle Ni-NTA resin by centrifugation at   and aspirate the supernatant using a gel loading tip to reduce the chance of removing Ni-NTA resin.
Centrifuge: 500 34, 1 min, 4 10
?. [Checking the Mass of Protein Bound to Resin]
Incubate Ni-NTA resin in 4x the resin volume of elution buffer for  at   with agitation.
4 °C
?. [Checking the Mass of Protein Bound to Resin]
Settle Ni-NTA resin by centrifugation at   and determine the supernatant protein concentration using a Bradford assay.
Centrifuge: 1000 33, 1 min
?. [Checking the Mass of Protein Bound to Resin]
Use the concentration to determine the protein mass bound to 50 ul Ni-NTA resin and upscale to calculate the total amount of protein bound to the Ni-NTA resin.
?. [Bradford Assay]
Dilute a   in deionised water to give a standard curve of 2 mg/ml, 1.5 mg/ml, 1.0 mg/ml, 0.75 mg/ml, 0.5 mg/ml, 0.25 mg/ml and 0.125 mg/ml.
[BSA stock solution]
?. [Bradford Assay]
Transfer two 1 ml aliquots of Bradford reagent per protein sample tested and BSA standard curve sample, and a single aliquot of 1 ml Bradford reagent to use as a blank to 2 ml cuvettes.
?. [Bradford Assay]
Add  to each Bradford assay aliquot, add  , or deionised water for samples diluted 5x or greater to the cuvette used as a blank.
[of the relevant samples]
[either elution buffer for undiluted samples]
?. [Bradford Assay]
Vortex solutions and incubate at room temperature for  and measure absorptions at 595 nm using spectrometer.
?. [Bradford Assay]
Plot the BSA standard curve, reading off the samples concentration if it is within the linear range of the curve.
?. [Bradford Assay]
If the sample is outside the linear range repeat the Bradford assay with the sample’s dilution adjusted to place it within the linear range.
?. [6His-SUMO Cleavage]
Add a 1:5 mass ratio of His-Senp1 : bound protein mass to the Ni-NTA resin, invert the resin to put into suspension and leave at   in a tube rack without agitation.
4 °C
?. [6His-SUMO Cleavage]
After an hour suspend the resin again and leave at  [  ] without agitation.
4 °C
?. [6His-SUMO Cleavage]
Transfer Ni-NTA resin to a gravity flow column and collect the flow through and wash the column in ¾ resin volume of cleavage buffer.
?. [6His-SUMO Cleavage]
Determine whether the protein has flowed through the column by comparing the colour change between  mixed with  and  mixed with  through at the point the buffer reaches the top of the resin bed.
[Bradford reagent]
[cleavage buffer]
[Bradford reagent]
[column flow]
?. [6His-SUMO Cleavage]
Add another ¾ resin volume of cleavage buffer if the flow through still has protein present.
?. [6His-SUMO Cleavage]
Once there is no protein in the flow through continue to size exclusion chromatography.
?. [Size exclusion chromatography]
Concentrate the eluted fraction to a volume of less than 1.5 ml by centrifuging at  at a time at   in a 10 kDa cut-off concentration column, mix the solution between each centrifugation to prevent the protein concentration getting too high at the base of the filter.
Centrifuge: 3000 34, 15 min, 10 10
10 °C
?. [Size exclusion chromatography]
Transfer the concentraed sample to a 1.5 ml centrifuge tube and centrifuge at  , this should leave a small pellet.
Centrifuge: 17000 34, 15 min
?. [Size exclusion chromatography]
Transfer the supernatant to a fresh 1.5 ml microcentrifuge tube and load into a 2 ml injection loop.
?. [Size exclusion chromatography]
Resolve on a superdex 200 16/600 column, the Parkin peak generally should elute at 85-95 ml, pS65 Parkin elutes later than wt Parkin.
?. [Size exclusion chromatography]
Pool fractions under the peak, taking only those close to the centre if very pure Parkin is required.
?. [Size exclusion chromatography]
Concentrate the pooled fractions to between 1 and 2 mg/ml using a 10 kDa cut off concentration column using a series of centrifugation pulses at  , mixing the sample after each pulse.
Centrifuge: 3000 33, 15 min, 10 10
?. [Size exclusion chromatography]
Aliquot the final protein sample, flash freeze in liquid nitrogen and store at   until required.
-80 °C
?. [Quality control]
Test protein purity using resolve  using a -  polyacrylamide gel using SDS-PAGE.
[final fraction]
?. [Quality control]
Observe Parkin and any possible contaminants by staining using a Coomassie brilliant blue stain.
?. [Quality control]
Test Parkin activity by performing a Parkin ubiquitination assay.
?. [Quality control]
Unphosphorylated wt Parkin should have no ubiquitination activity, however it should become active when treated with TcPINK1.
?. [Quality control]
It is possible to express Parkin with some activity, the suitability of this protein depends on the final application.