Oct 17, 2024
Expression and purification of LRRK2 constructs
- Verena Dederer1,2,
- Deep Chatterjee1,2,
- Stefan Knapp1,2,
- Sebastian Mathea1,2
- 1Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Straße 9, Frankfurt 60438, Germany;
- 2Structural Genomics Consortium, Buchman Institute for Molecular Life Science (BMLS), Max-von-Laue-Straße 15, Frankfurt 60438, Germany
Protocol Citation: Verena Dederer, Deep Chatterjee, Stefan Knapp, Sebastian Mathea 2024. Expression and purification of LRRK2 constructs. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn314pl5d/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: January 05, 2024
Last Modified: October 17, 2024
Protocol Integer ID: 92975
Abstract
Expression and purification of human LRRK2 and its variants from insect cells.
Guidelines
if not otherwise mentioned every step was performed on ice.
Materials
Equipment
Centrifuge: Thermo Scientific Sorvall LYNX 6000
AKTA: ÄKTA start; ÄKTA Pure 25
Ni-NTA (Cytiva #17531803)
SP-sepharose column (Cytiva #17505701)
HiLoad 16/600 Superdex 200 pg gel filtration column
Amicon Ultra 15 mL Centrifugal Filters 10,000 MWCO (Millipore)
Buffers
Lysis buffer: 50 mM HEPES 7.4 , 500 mM NaCl, 20 mM imidazole, 5 mM MgCl2, 20 µM GDP, 0.5 mM TCEP, 5% glycerol
Ni-NTA elution buffer: 50 mM HEPES 7.4, 500 mM NaCl, 300 mM imidazole, 5 mM MgCl2, 20 µM GDP, 0.5 mM TCEP, 5% glycerol
no salt buffer: 20 mM HEPES 7.4, 5 mM MgCl2, 20 µM GDP, 0.5 mM TCEP, 5% glycerol
high salt buffer: 20 mM HEPES 7.4, 2.5 M NaCl, 5 mM MgCl2, 20 µM GDP, 0.5 mM TCEP, 5% glycerol
gel filtration buffer: 20 mM HEPES 7.4, 150 mM NaCl, 5 mM MgCl2, 20 µM GDP, 0.5 mM TCEP, 0.5% glycerol
Safety warnings
All experiments were performed under the rules of S1 lab regulations.
Before start
Buffers, beads, columns are chillled at 4°C prior to use.
Protein expression and purification
Protein expression and purification
2d 18h
2d 18h
exponentially growing SF9 cells (2x10^6 cells/mL in Lonza Insect-XPRESS medium) are transduced with high-titre bacculovirus suspension (encoding TEV cleavable N-terminally His6-Z-tagged human LRRK2).
After shaking with90 rpm at27 °C for 66:00:00 , cells are harvested by centrifugation.
2d 18h
Pellets are washed once with PBS and frozen until further use.
Cell pellets are resuspended in lysis buffer and lysed by sonication on ice using a 13-mm probe (35% amplitude, 5 sec pulse, 10 sec pause, 5 min total pulse time).
Lysate is cleared by ultracentrifugation for 1 hour with 100.000 x g, 4°C
Clarified lysate is loaded onto pre-equilibrated Ni-NTA in gravity flow columns.
After extensive washing with lysis buffer (20 CV), the His-tagged protein is eluted with elution buffer containing 0.300 Molarity (M) Imidazole
After dilution of the eluate's salt concentration to 250 millimolar (mM) NaCl using no salt buffer, the protein is loaded onto a SP sepharose column connected to an Äkta Start system (Cytiva).
After washing with buffer containing 250 millimolar (mM) NaCl the bound LRRK2 construct is eluted with a salt gradient ranging from 250 mM to 2.5 M NaCl while elution fractions are collected throughout the run.
Peak fractions are pooled and TEV protease (molar ratio of 1:100) is added during overnight incubation rolling at 4 °C .
Cleaved tag, TEV protease and other contaminating proteins are removed by an combined SP sephareose-Ni-NTA rebinding.
LRRK2 construct is concentrated and subjected to gel filtration in gel filtration buffer on a HiLoad 16/600 S200 column using an Äkta Pure system.
Peak fractions are combined, the protein is concentrated.
the UV absorbance is measure and the protein is flash frozen in liquid nitrogen for storage.