Jul 13, 2023
LRRK1 expression and purification
- Yu Xuan Lin1,2,
- janice reimer1,2
- 1Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
Protocol Citation: Yu Xuan Lin, janice reimer 2023. LRRK1 expression and purification. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzx3b5gx1/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: July 10, 2023
Last Modified: July 13, 2023
Protocol Integer ID: 84802
Funders Acknowledgement:
Aligning Science Across Parkinson's: ASAP
Abstract
Protein purification protocol for full-length LRRK1 as done by Leschziner and Reck-Peterson Labs.
Original protocol by Janice M. Reimer and Yu Xuan Lin.
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
His6-Z-TEV-LRRK1 Purification
His6-Z-TEV-LRRK1 Purification
4d 6h
4d 6h
N-terminally tagged His6-Z-TEV-LRRK1(FL) was expressed in Sf9 insect cells. Insect cells were infected with baculovirus and grown at 27°C for 3 days.
3d
Cells were harvested and then resuspended in lysis buffer (50mM HEPES pH 7.4, 500 mM NaCl, 20 mM imidazole, 0.5 mM TCEP, 5% glycerol, 5 mM MgCl2, 20 μM GDP, 0.5 mM Pefabloc and cOmplete EDTA-free protease inhibitor cocktail (Roche).
1h
Cells were lysed using a Dounce homogenizer and clarified by centrifugation at 50000 rpm, 4°C, 01:28:00, and Ti70 rotor.
2h
The supernatant was incubated for 1 hr with Ni-NTA agarose beads (Qiagen) equilibrated in lysis buffer. Beads were applied to a gravity column where they were extensively washed with lysis buffer, wash buffer ( 50mM HEPES pH 7.4, 500mM NaCl, 0.5mM TCEP, 5% glycerol, 5mM MgCL2, 20uM GDP), followed by elution in lysis buffer containing 300 mM imidazole ( 50mM HEPES pH 7.4, 500mM NaCl, 0.5mM TCEP, 5% glycerol, 5mM MgCL2, 20uM GDP, 300mM imidazole)
2h
The eluted protein was diluted to 250 mM NaCl using dilution buffer (50mM HEPES pH 7.4, 0.5mM TCEP, 5% glycerol, 5mM MgCL2, 20uM GDP) and loaded onto a SP Sepharose column (Cytiva) equilibrated in buffer (50 mM HEPES pH 7.4, 250 mM NaCl, 0.5 mM TCEP, 5% glycerol, 5 mM MgCl2, 20 μM GDP)
1h
The protein was eluted using a 250 mM to 2.5 M NaCl gradient. Fractions containing LRRK1(FL) were pooled, diluted to ~500 mM NaCl, and incubated with TEV protease overnight at 4°C.
18h
The protein was concentrated and put directly over a S200 size exclusion column (Cytiva) equilibrated in storage buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 5% glycerol, 5 mM MgCl2 and 20 μM GDP).
5h
The protein was concentrated to ~5-6 μM and flash frozen in liquid nitrogen for storage. N-terminally tagged His6-Z-TEV-LRRK120-2015 was purified using the same protocol.
1h
Protocol references