TITLE: Rab8a expression and purification
AUTHORS: David M. Snead, Mariusz  Matyszewski

[DESCRIPTION]
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.

[STEPS]
SECTION: Expression
1. Transform n-terminally His6-ZZ tagged Rab8a (pET28a backbone) into BL21(DE3) E. coli cells.

 




SECTION: Expression
3. Grow an overnight culture in LB media with 50 µg/mL 
Make 50 mL . This is enough for the main 4L growth.

SECTION: Expression
4. Add the overnight culture into main LB flasks with antibiotic present (50 µg/mL ). Use 100x dilution (10 mL ).

SECTION: Expression
2. Make fresh LB media. This protocol assumes 4 L are made. Scale accordingly.

SECTION: Expression
5. Let it growth in a shaker at   until OD600 of 0.4-0.5 is reached.

SECTION: Expression
6. Chill the cells and set the shaker to 18 °C . We chill the cells in the cold room at 4 °C for 60 min 

SECTION: Expression
7. Add IPTG at 0.5 millimolar (mM)  into the chilled culture.

 


SECTION: Expression
8. Let it grow in a shaker at   

SECTION: Expression
9. Harvest the cells. We spun them down using a JLA 9.1000 rotor at6000 rpm, 6 min, 4 °C in 1L Beckman bottles. We then resuspended the cells in 15-30 mL and transferred them to 50 mL Falcon tubes (1 tube per 2L harvested). Flash frozen with liquid nitrogen and stored in -80 °C freezer.

SECTION: Purification: Day 1
10. Resuspend the cell pellet in lysis buffer. Add about 40 mg  and incubate 30 min at on ice 

SECTION: Purification: Day 1
11. Lyse the cells by sonication on ice .

SECTION: Purification: Day 1
12. Ultracentrigation in Ti-70 rotor 30000 rpm, 30 min, 4 °C 

 

SECTION: Purification: Day 1
13. Once centrifugation is done, add the beads to the supernatant. Let it nutate for 60 min 4 °C .

SECTION: Purification: Day 1
12.1. While centrifuging, prepare 2 mL by equilibrating with lysis buffer.

SECTION: Purification: Day 1
13.1. Prepare Wash and Elution buffers while waiting.
SECTION: Purification: Day 1
14. Apply beads to a gravity column and wash with 250 mL 

SECTION: Purification: Day 1
15. Elute with 40 mL . Best to resuspend in 20 mL , elute, resuspend again, and finish elution.

SECTION: Purification: Day 1
16. Equilibrate IgG beads with Wash Buffer.
SECTION: Purification: Day 1
17. Dilute the eluted protein to 90 mL . Split in 2 (45 mL ). Add 2 mL  to each. 

SECTION: Purification: Day 1
18. Nutate at 4 °C  for 2-3 hours.

SECTION: Purification: Day 1
19. Apply to gravity column and wash with 250 mL 

SECTION: Purification: Day 1
20. Make TEV buffer and transfer beads with 10 mL into 15 mL tubes.

SECTION: Purification: Day 1
21. Add 400 µL 1.3 mg/mL TEV protease and incubate   at 4 °C 

SECTION: Purification: Day 2
22. Equilibrate Ni-NTA beads with TEV buffer in gravity column.
SECTION: Purification: Day 2
23. Pour cleaved protein onto the beads. Collect flowthrough.
SECTION: Purification: Day 2
24. Wash with leftover TEV buffer. Collect flowthrough as well.
SECTION: Purification: Day 2
25. Combine all flowthroughs and concentrate to 1 mL 

SECTION: Purification: Day 2
26. Equilibrate S200I column with S200 buffer.

 

SECTION: Purification: Day 2
27. Apply concentrated protein to the S200I column and run S200I program.
SECTION: Purification: Day 2
28. Concentrate protein and do buffer exchange with TEV buffer modified with 10% glycerol.
SECTION: Purification: Day 2
29. Concentrate protein, quantify, and flash freeze for -80 °C storage.