Apr 01, 2025
PPM1H Purification from E. coli
- 1Stanford University;
- 2Stanford University School of Medicine
Protocol Citation: Ayan Adhikari, Suzanne R Pfeffer 2025. PPM1H Purification from E. coli. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbnp9ygpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 01, 2025
Last Modified: April 01, 2025
Protocol Integer ID: 125909
Keywords: ASAPCRN, LRRK2, Rab GTPase, Rab phosphorylation, Rab phosphatase, Parkinson's
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000463
Abstract
This protocol is used to purify full length PPM1H enzyme in good yield and purity after expression in bacteria. We generally obtain 2mg from 4L bacterial culture and express the protein at 18°C to avoid aggregation due to overexpression.
Materials
Lysis Buffer:
- 50mM HEPES pH 8
- 500mM NaCl
- 5mM MgCl2
- 0.5 mM TCEP
- EDTA free Protease tablet (1/50mL)
- 10% glycerol
Wash Buffer:
- 50mM HEPES pH 8
- 500mM NaCl
- 5mM MgCl2
- 20mM imidazole
- 0.5 mM TCEP
- 10% glycerol
Elution Buffer:
- 50mM HEPES pH 8
- 500mM NaCl
- 5mM MgCl2
- 500mM imidazole
- 0.5 mM TCEP
- 10% glycerol
Size Exclusion/Dialysis Buffer:
- 50mM HEPES pH 8
- 150mM NaCl
- 5mM MgCl2
- 0.5 mM TCEP
- 10% glycerol
LB agar plates containing 100µg/ml carbenicillen and 34µg/ml chloramphenicol
Per flask, 2ml each of 1000X carbenicillin (100mg/ml in H2O) and chloramphenicol (34mg/ml in EtOH)
Roche EDTA-free Protease inhibitor tablets
Avestin Emulsiflex unit for cell breakage
SUMO protease (Ulp1)
Day 1: Prepare a Starter Culture and Sterilize media
Day 1: Prepare a Starter Culture and Sterilize media
Pick a fresh colony from an LB agar plate to inoculate 50 mL LB broth. Grow Overnight with rotation at 37 °C
Autoclave 2 x 6L flasks, each containing 2 L LB broth. Store at 37 °C for inoculation the following day
Day 2: Inoculation and Induction
Day 2: Inoculation and Induction
Inoculate each 6L flask with 20 mL of starter culture
Add 2 mL , 1000X Carbenicillin and 2 mL , 1000X chloramphenicol to flask and rotate at 37 °C until OD600 0.5-0.6 is reached
Take an initial OD measurement 1hr post inoculation
Doubling time is 20-30min. Calculate how long to reach 0.6 (usually 2.5 hr)
Optional: use remaining O/N culture to make additional bacterial glycerol stocks
Glycerol stocks are made using 500 µL of bacterial culture and 500 µL of 50% Glycerol
Transfer to -80 °C immediately
Once OD is reached, transfer flasks to 18 °C controlled temperature shaker and equilibrate temperature for 10-20min
Add IPTG to 0.3 millimolar (mM)
Incubate with rotation overnight (180 RPM) at 18 °C
Day 3: Purification Protocol
Day 3: Purification Protocol
Resuspension and Lysis
Transfer cultures to 1L centrifuge bottles
Pellet bacteria by spinning at 4000 RPM in a Sorvall centrifuge for 00:20:00 at 4 °C ; discard supernatant
20m
Add 1 Roche protease inhibitor tablet to 50mL of lysis buffer, rotate ~10 min at 4 °C to dissolve
Set up a ~200mL beaker with a magnetic stir bar at 4 °C
Resuspend pelleted bacteria on ice or in cold room using ~10 mL lysis buffer per bottle
Vortex the bottles to resuspend the pellet
Transfer slurry to 200ml beaker with stir bar
Pass 20 mL lysis buffer through the Emulsiflex at 0-30K PSI to equilibrate machine
Pass bacterial slurry through Emulsiflex once at 40-60K PSI to lyse cells. Collect into Oakridge tube(s)
If the slurry resists pipetting up and down, add additional lysis buffer (no more than 20 mL more)
Balance tubes
Centrifuge at 40,000 RPM for 45min in a 45Ti rotor at 4 °C to pellet cell debris
Collect supernatant and pass through a 500ml, 0.45µm Millipore filter under vacuum. Transfer the clarified supernatant into a 50ml glass beaker
HiTrap Talon (Cobalt) Affinity Purification using FPLC
Wash a 1mL HiTrap Talon column with 10 column volumes (CV) of distilled degassed H2O
Equilibrate column with 10CV of lysis buffer
Pass the lysate through the column. Collect the flow through
Wash the column with 30CV of wash buffer. Collect the wash fractions
Elute using an imidazole gradient of 100mM-500mM. Collect the elution fractions and analyze by SDS PAGE
Pool the eluted fractions containing the desired protein and dialyze overnight into dialysis buffer in the presence of SUMO protease (Ulp1) (100ng/1mg of protein). We usually dialyze ~6mL
Collect the dialyzed protein the next day and concentrate it by microfiltration on an Centricon 10K cutoff filter before resolving the protein on a S200 24 ml column fitted in series with a 1ml HiTrap Talon column to remove uncleaved His SUMO protein and the His-tagged SUMO protease
Collect 4-5, 4ml fractions. Analyze by SDS-PAGE and pool the fractions containing the desired protein
Concentrate the protein by Centricon filtration if needed
Snap freeze in liquid nitrogen and store at -80 °C
Shown below are SDS-PAGE gels of the initial Cobalt column and gel filtration fractions after His-SUMO tag removal
SDS-PAGE of column fractions from this protocol; yield is ~2mg/4L culture
Protocol references
Yeshaw WM, Adhikari A, Chiang CY, Dhekne HS, Wawro PS, Pfeffer SR. Localization of PPM1H phosphatase tunes Parkinson's disease-linked LRRK2 kinase-mediated Rab GTPase phosphorylation and ciliogenesis. Proc Natl Acad Sci U S A. 2023 Oct 31;120(44):e2315171120. doi: 10.1073/pnas.2315171120.
PMID: 37889931; PMCID: PMC10622911.