May 23, 2022
Rab8a expression and purification
- David M. Snead1,2,
- 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 M. Snead, Mariusz Matyszewski 2022. Rab8a expression and purification. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr63mzvmk/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: March 03, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 59034
Keywords: Rab8a, protein purification, protein expression, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000519
MJFF
Grant ID: 18321
Abstract
Recombinant Rab8a expression and purification protocol as used by the Leschziner and Reck-Peterson Labs.
Original protocol by David Snead. Adapted for protocols.io by Mariusz Matyszewski.
Current version as used in Snead, Matyszewski, Dickey et al. 2022.
Materials
Materials:
Ni-NTA beads
IgG beads
Purified TEV protease
Equipment:
FPLC
Ultracentrifuge
Sonicator
Buffers:
Lysis Buffer (make 150 mL ):
- 50 millimolar (mM) HEPES pH 7.4
- 200 millimolar (mM) NaCl
- 10 % volume Glycerol
- 5 millimolar (mM) MgCl2
- 2 millimolar (mM) DTT
- 0.5 millimolar (mM) PefaBloc
- 1 cOmplete Protease Inhibitor Cocktail tablet per 50 mL (3 for suggested volume in this prep)
Wash Buffer (make 750 mL ):
- 50 millimolar (mM) HEPES pH 7.4
- 150 millimolar (mM) NaCl
- 5 % volume Glycerol
- 5 millimolar (mM) MgCl2
- 1 millimolar (mM) DTT
Elution Buffer (make 100 mL ):
- 50 millimolar (mM) HEPES pH 7.4
- 150 millimolar (mM) NaCl
- 5 % volume Glycerol
- 5 millimolar (mM) MgCl2
- 1 millimolar (mM) DTT
- 300 millimolar (mM) Imidazole pH 8.0
TEV Buffer (make 200 mL ; will also need a little bit of extra TEV buffer with 10 % volume Glycerol for storage):
- 50 millimolar (mM) HEPES pH 7.4
- 200 millimolar (mM) NaCl
- 5 % volume Glycerol
- 5 millimolar (mM) MgCl2
- 1 millimolar (mM) DTT
S200 Buffer (make 250 mL ):
- 50 millimolar (mM) HEPES pH 7.4
- 200 millimolar (mM) NaCl
- 1 % volume Glycerol
- 5 millimolar (mM) MgCl2
- 1 millimolar (mM) DTT
Expression
Expression
1h 6m
1h 6m
Transform n-terminally His6-ZZ tagged Rab8a (pET28a backbone) into BL21(DE3) E. coli cells.
Note
BL21-CodonPlus (DE3)-RIPL have also been used successfully. Please adjust the antibiotics used based on the cells used.
pET28a backbone comes with Kanamycin resistance
Make fresh LB media. This protocol assumes 4 L (2L per flask) are made. Scale accordingly.
Grow an overnight culture in LB media with 50 µg/mL Kanamycin
Make 50 mL . This is enough for the main 4L growth.
Add the overnight culture into main LB flasks with antibiotic present (50 µg/mL Kanamycin ). Use 100x dilution (10 mL per 1L ).
Let it growth in a shaker at 200 rpm, 37°C until OD600 of 0.4-0.5 is reached.
Chill the cells and set the shaker to 18 °C . We chill the cells in the cold room at 4 °C for 01:00:00
1h
Add IPTG at 0.5 millimolar (mM) final concentration into the chilled culture.
Note
Higher IPTG concentrations can be used if needed.
Let it grow in a shaker at 200 rpm, 18°C, 18:00:00
Harvest the cells. We spun them down using a JLA 9.1000 rotor at6000 rpm, 4°C, 00:06:00 in 1L Beckman bottles. We then resuspended the cells in 15-30 mL LB and transferred them to 50 mL Falcon tubes (1 tube per 2L harvested). Flash frozen with liquid nitrogen and stored in -80 °C freezer.
6m
Purification: Day 1
Purification: Day 1
2h
2h
Resuspend the cell pellet in lysis buffer. Add about 40 mg Lysozyme and incubate 00:30:00 at On ice
30m
Lyse the cells by sonication On ice .
Ultracentrigation in Ti-70 rotor 30000 rpm, 4°C, 00:30:00
Note
Done using ultracentrifugation in our lab, but regular centrifugation should work too. Adjust accordingly.
30m
While centrifuging, prepare 2 mL Ni-NTA beads by equilibrating with lysis buffer.
Once centrifugation is done, add the beads to the supernatant. Let it nutate for 01:00:00 4 °C .
1h
Prepare Wash and Elution buffers while waiting.
Apply beads to a gravity column and wash with 250 mL Wash buffer
Elute with 40 mL Elution buffer . Best to resuspend in 20 mL , elute, resuspend again, and finish elution.
Equilibrate IgG beads with Wash Buffer.
Dilute the eluted protein to 90 mL Wash Buffer . Split in 2 (45 mL each ). Add 2 mL IgG beads to each.
Nutate at 4 °C for 2-3 hours.
Apply to gravity column and wash with 250 mL Wash buffer
Make TEV buffer and transfer beads with 10 mL TEV Buffer into 15 mL tubes.
Add 400 µL 1.3 mg/mL TEV protease and incubate Overnight at 4 °C
Purification: Day 2
Purification: Day 2
Equilibrate Ni-NTA beads with TEV buffer in gravity column.
Pour cleaved protein onto the beads. Collect flowthrough.
Wash with leftover TEV buffer. Collect flowthrough as well.
Combine all flowthroughs and concentrate to 1 mL
Equilibrate S200I column with S200 buffer.
Note
Other SEC columns can be used instead. S75 should have good separation too.
Apply concentrated protein to the S200I column and run S200I program.
Concentrate protein and do buffer exchange with TEV buffer modified with 10% glycerol.
Concentrate protein, quantify, and flash freeze for -80 °C storage.