Aug 25, 2023
LRRK2 expression and purification
- Xinbo Wang1,2,
- Pietro De Camilli1,2
- 11. Departments of Neuroscience and of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 22.Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Protocol Citation: Xinbo Wang, Pietro De Camilli 2023. LRRK2 expression and purification. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv59wd4g1b/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: August 19, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 68877
Keywords: LRRK2, Expi293F cells, In vitro purification, ASAPCRN
Abstract
This protocol details methods for the expression of human LRRK2 in Expi293F cells and its in vitro purification.
Attachments
iut8bvk9p.docx
20KB
Materials
ExpiFectamine™ 293 Transfection KitThermo FisherCatalog #A14525
Prescission ProteaseGenscriptCatalog #Z02799
3xFLAG PeptideSigma – AldrichCatalog #F4799
cOmplete™, EDTA-free Protease Inhibitor CocktailSigma AldrichCatalog #05056489001
Glutathione Sepharose (GE Healthcare, 17075601)
Slide-A-Lyzer™ MINI Dialysis Device, 10K MWCO, 0.1 mLThermo FisherCatalog #69572
Amicon Ultra-15 Centrifugal Filter Unit Millipore SigmaCatalog #UFC901024 & UFC903024
Monoclonal ANTI-FLAG M2 resinMillipore SigmaCatalog #F3165
EDTA-free protease inhibitor cocktail (Roche).
Solutions to prepare:
Lysis salt buffer:
| A | B | |
| HEPES (7.4) | 20 mM | |
| NaCl | 500 mM | |
| Glycerol | 10 % | |
| DTT | 2 mM | |
| 1xcomplete EDTA-free protease inhibitor |
Dialysis buffer:
| A | B | |
| HEPES (7.4) | 20 mM | |
| NaCl | 150 mM | |
| Glycerol | 5 % | |
| MgCl2 | 2.5 mM | |
| DTT | 2 mM | |
| GDP | 20 μM |
LRRK2 expression and purification
LRRK2 expression and purification
3h 14m
3h 14m
Transfect the constructs encoding 3xFlag-LRRK2, 3xFlag-LRRK2(I2020T), 3xFlag-RCKW or 3xFlag-GFP-LRRK2 into Expi293F cells according to manufacturer instructions.
Express the proteins for 72:00:00 following induction according to manufacturer instructions.
3d
Harvest the cells by centrifugation (400 x g , 00:04:00 ) and lyse by 3 freeze-thaw cycles in lysis buffer.
Note
Note: For 60 mL of cell suspension, we used 15 mL lysis buffer.
4m
Remove the cellular debris by centrifugation at 15000 x g for 01:00:00 at 4 °C .
1h
Mix the clarified lysate with anti-FLAG M2 resin for 02:00:00 while rotating at 4 °C .
Note
Note: For 60 mL of cell suspension, we used 180 µL of Anti-FLAG resin.
2h
Wash the resin with 3x10 bed volumes of lysis buffer.
Elute the proteins with lysis buffer supplemented with 0.2 mg/mL 3xFlag peptides.
Note
Note: For 60 mL of cell suspension, we used 800 µL elution buffer (without protease inhibitor).
Remove the N-terminal 3xFlag tag by incubation with the GST tagged Prescission Protease (0.01 U/µL ) Overnight while rotating at 4 °C .
Remove the GST tagged Prescission Protease subsequently by Glutathione Sepharose.
Assess the purity of the proteins by SDS-PAGE and Western blotting.
Dialyze the purified proteins Overnight at 4 °C against the dialysis buffer.
After dialysis, clarify the proteins by centrifugation at 17000 x g for 00:10:00 at 4 °C .
10m
Determine the protein concentration by SDS-PAGE using Bovine Serum Albumin (BSA) as standard and used without freezing in liposome binding and tubulation experiments.