Rab8a expression and purification
David M. Snead, Mariusz Matyszewski
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.
Steps
Expression
Transform n-terminally His6-ZZ tagged Rab8a (pET28a backbone) into BL21(DE3) E. coli cells.
Make fresh LB media. This protocol assumes 4L are made. Scale accordingly.
Grow an overnight culture in LB media with 50µg/mL
Make 50mL . This is enough for the main 4L growth.
Add the overnight culture into main LB flasks with antibiotic present (50µg/mL ). Use 100x dilution (10mL ).
Let it growth in a shaker at 200rpm 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 1h 0m 0s
Add IPTG at 0.5millimolar (mM) into the chilled culture.
Let it grow in a shaker at 200rpm
Harvest the cells. We spun them down using a JLA 9.1000 rotor at6000rpm,4°C in 1L Beckman bottles. We then resuspended the cells in 15-30mL and transferred them to 50 mL Falcon tubes (1 tube per 2L harvested). Flash frozen with liquid nitrogen and stored in -80°C freezer.
Purification: Day 1
Resuspend the cell pellet in lysis buffer . Add about 40mg and incubate 0h 30m 0s at 4On ice
Lyse the cells by sonication On ice .
Ultracentrigation in Ti-70 rotor 30000rpm,4°C
While centrifuging, prepare 2mL by equilibrating with lysis buffer .
Once centrifugation is done, add the beads to the supernatant. Let it nutate for 1h 0m 0s 4°C .
Prepare Wash and Elution buffers while waiting.
Apply beads to a gravity column and wash with 250mL
Elute with 40mL . Best to resuspend in 20mL , elute, resuspend again, and finish elution.
Equilibrate IgG beads with Wash Buffer .
Dilute the eluted protein to 90mL . Split in 2 (45mL ). Add 2mL to each.
Nutate at 4°C for 2-3 hours.
Apply to gravity column and wash with 250mL
Make TEV buffer and transfer beads with 10mL into 15 mL tubes.
Add 400µL 1.3mg/mL TEV protease and incubate at 4°C
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 1mL
Equilibrate S200I column with S200 buffer .
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.
