May 23, 2022
LRRK2 RCKW Protein Purification
- David Snead1,2,
- Yu Xuan Lin1,
- Mariusz Matyszewski1
- 1Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093;
- 2Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Heath, Baltimore, MD, 21205
Protocol Citation: David Snead, Yu Xuan Lin, Mariusz Matyszewski 2022. LRRK2 RCKW Protein Purification. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgb6693lpk/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: February 09, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 57957
Keywords: LRRK2, protein purification, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000519
MJFF
Grant ID: 18321
Abstract
Protein purification protocol for tag-less LRRK2RCKW as done by Leschziner and Reck-Peterson Labs. Same protocol can be used to purify LRRK1RCKW as well.
Original Protocol by David Snead. Modified by Yu Xuan Lin and Mariusz Matyszewski for publication.
Materials
Equipment:
Ultracentrifuge, we used one with a Ti70 rotor
Dounce homogenizer
FPLC with SP, His, and S200I columns.
Also make sure Ni-NTA beads and TEV protease are available.
Buffers (suggested volumes listed):
Day 1:
300 mL Lysis Buffer :
50 millimolar (mM) HEPES 7.4
0.5 Molarity (M) NaCl
20 millimolar (mM) Imidazole 8.0
0.5 millimolar (mM) TCEP
5 % volume Glycerol
5 millimolar (mM) MgCl2
20 micromolar (µM) GDP
Plus protease inhibitors (Protein inhibitor tablets and 0.5 millimolar (mM) Pefabloc )
300 mL Wash Buffer :
50 millimolar (mM) HEPES 7.4
0.5 Molarity (M) NaCl
0.5 millimolar (mM) TCEP
5 % volume Glycerol
5 millimolar (mM) MgCl2
20 micromolar (µM) GDP
100 mL Elution Buffer :
50 millimolar (mM) HEPES 7.4
0.5 Molarity (M) NaCl
300 millimolar (mM) Imidazole 8.0
0.5 millimolar (mM) TCEP
5 % volume Glycerol
5 millimolar (mM) MgCl2
20 micromolar (µM) GDP
300 mL Dilution Buffer :
50 millimolar (mM) HEPES 7.4
0.5 millimolar (mM) TCEP
5 % volume Glycerol
5 millimolar (mM) MgCl2
20 micromolar (µM) GDP
500 mL Buffer A (250 mM NaCl) :
50 millimolar (mM) HEPES 7.4
250 millimolar (mM) NaCl
0.5 millimolar (mM) TCEP
5 % volume Glycerol
5 millimolar (mM) MgCl2
20 micromolar (µM) GDP
300 mL Buffer B (2.5 M NaCl) :
50 millimolar (mM) HEPES 7.4
2.5 Molarity (M) NaCl
0.5 millimolar (mM) TCEP
5 % volume Glycerol
5 millimolar (mM) MgCl2
20 micromolar (µM) GDP
Day 2:
500 mL Buffer 0 :
50 millimolar (mM) HEPES 7.4
0.5 Molarity (M) NaCl
0.5 millimolar (mM) TCEP
5 % volume Glycerol
5 millimolar (mM) MgCl2
20 micromolar (µM) GDP
300 mL Buffer 300 :
50 millimolar (mM) HEPES 7.4
0.5 Molarity (M) NaCl
300 millimolar (mM) Imidazole 8.0
0.5 millimolar (mM) TCEP
5 % volume Glycerol
5 millimolar (mM) MgCl2
20 micromolar (µM) GDP
300 mL Storage Buffer :
20 millimolar (mM) HEPES 7.4
0.7 Molarity (M) NaCl
0.5 millimolar (mM) TCEP
5 % volume Glycerol
2.5 millimolar (mM) MgCl2
20 micromolar (µM) GDP
Day 1: Protein Pellet Lysis
Day 1: Protein Pellet Lysis
1h 28m
1h 28m
Ready the Ultracentrifuge and cool it down to 4 °C
Make Lysis Buffer (check Materials section)
Thaw protein pellets On ice
Resuspend each pellet in 40 mL Lysis Buffer
Note
Might need to use a spatula and a pipette to resuspend fully.
Homogenize each pellet with Dounce homogenizer On ice
15 plunges loose, followed by 17 plunges tight.
Note
Gentle when homogenizing, don’t completely pull douncer out of liquid, avoid bubbles
Pour lysate into rotor tubes (pre-chilled, clean, check for cracks; we used Ti70 tubes)
Make sure they are balanced. Use Lysis buffer to balance if needed.
Spin lysate in ultracentrifuge 50000 rpm, 4°C, 01:28:00 , Ti70 rotor
1h 28m
Prep the rest of Day 1 buffers while waiting
Day 1: Ni-NTA gravity column
Day 1: Ni-NTA gravity column
2m
2m
Start equilibrating 12 mL of Ni-NTA beads with Lysis buffer when there is about 30 mins left on the centrifuge
We equilibrated by resuspending 6 mL in a 15 mL falcon tube, twice.
They were spun down at 1000 rpm, 4°C, 00:02:00 , 3 times , each time getting rid of liquid and doing a 50:50 resuspension.
2m
After ultracentrifuge is done, pour the supernate into 3x 50 mL falcon tubes.
Add 4 mL equilibrated Ni-NTA beads to 40 mL of supernate. Bring up to 50 mL with Lysis buffer
Incubate in cold room, while rotating "hot-dog style". 0 rpm, 4°C, 01:00:00 medium-slow rotation
After incubation, add onto a gravity column. Wash with remaining Lysis Buffer, followed by 200 mL Wash Buffer
Elute with 50 mL Elution Buffer
Make sure to resuspend the beads with stopper closed on the column. Might need multiple repeats of resuspension for best results. Leave column dry afterwards.
Dilute the solution to 250 millimolar (mM) NaCl by adding 50 mL Dilution Buffer
Syringe filter the solution.
Day 1: FPLC Cation Exchange and Overnight TEV cleavage
Day 1: FPLC Cation Exchange and Overnight TEV cleavage
Use a 5 mL HiTrap SP FF column. Pump wash FPLC with Buffer A and Buffer B. Equilibrate the column with Buffer A.
Apply sample onto the column with a sample pump
Run a gradient program, 250 millimolar (mM) NaCl to 2.5 Molarity (M) NaCl , 0% B to 100% B.
Combine protein fractions into a 50 mL Falcon Tube. Check protein concentration; should be around 1.1 micromolar (µM)
Afterwards dilute to 600 millimolar (mM) NaCl
Bring up to 50 mL with Wash Buffer
Add TEV, and incubate overnight at 4 °C while rotating "hot-dog style".
Note
Our final TEV concentration was around 0.2 micromolar (µM)
Day 2: FPLC HisTrap Column
Day 2: FPLC HisTrap Column
Make Day 2 buffers. Make sure to degas the Storage Buffer.
Put the 5 mL HisTrap column on the FPLC. Pump wash the FPLC with Buffer 0 and Buffer 300. Equilibrate the column with Buffer 0.
Run a Step Gradient Program
Note
0% for 4 CV
6.7% for 5 CV
13.3% for 5 CV
26.7% for 5 CV
50% for 4 CV
100% for 6 CV (Column Wash)
0% for 5 CV (Column Re-equilibration)
Combine protein elution into a 15 mL tube before concentrating for Size Exclusion Column
Day 2: SEC Column
Day 2: SEC Column
2m
2m
Equilibrate a S200I 10/300 column with the Storage Buffer.
Concentrate the protein to 1 mL using 100 kD cutoff concentrators. Should be done nearing the end of the column equilibration.
Note
We concentrated using 2 concentrators to save time.
After concentrating, filter the protein using a 0.1 uM spin filter. Spin at max speed for 00:02:00 at 4 °C
2m
Inject filtered sample and run elution program.
Collect and concentrate the protein to about 500 µL . Concentration should be around 25-30 micromolar (µM) after concentrating.
Note
About 3-6 mL of protein initially.
Aliquot and freeze protein. Make sure to check pureness of protein as well.
Note
2, 5, and/or 10 uL aliquots recommended.