TITLE: Production of GTPase Deficient RAB1A(Q70L) Protein
AUTHORS: Annan SI Cook

[DESCRIPTION]
Bacterial expression of the GTPase deficient Q70L mutant of human RAB1A.

[STEPS]
SECTION: Transformation and Colony Selection
1. Transform E. coli BL21 cells with the pET vector containing 6x-His-RAB1A(Q70L) by heat shock.
SECTION: Transformation and Colony Selection
2. Plate the transformed cells on ampicillin-resistant plates.
SECTION: Transformation and Colony Selection
3. Incubate the plates   at 37 °C.
SECTION: Pre-culture and Expansion
4. Inoculate a single colony into 10 mL of LB broth.

SECTION: Pre-culture and Expansion
5. Incubate the culture   at 37 °C.

SECTION: Main Culture Preparation
6. Transfer the entire pre-culture into a 1 L culture flask.
SECTION: Main Culture Preparation
7. Grow the 1 L culture at 37 °C with shaking at   until the optical density (OD) reaches approximately 0.6.

SECTION: Cooling and Induction
8. Place the flask in an ice bath for 15 min to cool rapidly.

SECTION: Cooling and Induction
9. Induce protein expression by adding 0.1 millimolar (mM) IPTG.

SECTION: Cooling and Induction
10. Continue incubating the culture   (approximately 16 hours) at 18 °C with shaking at  .
SECTION: Cell Harvest and Pelleting
11. Harvest the cells by centrifugation at 4000 rpm in a BECKMAN Coulter centrifuge.

SECTION: Cell Washing and Freezing
12. Wash the cell pellet once with PBS.
SECTION: Cell Washing and Freezing
13. Pellet the cells again at 4000 x g.

SECTION: Cell Washing and Freezing
14. Flash freeze the pellet in liquid nitrogen.
SECTION: Cell Washing and Freezing
15. Store the frozen pellet at -80 °C until purification.

SECTION: Cell Lysis
16. Thaw the frozen cell pellet to Room temperature.

SECTION: Cell Lysis
17. Resuspend the cells in lysis buffer.

Lysis buffer 
      HEPES pH 7.5  25 mM    NaCl  300 mM    MgCl2  2 mM    TCEP  1 mM  

SECTION: Cell Lysis
18. Add an EDTA-free protease inhibitor tablet (Thermo Fisher) and PMSF to a concentration of 1 millimolar (mM).
SECTION: Cell Lysis
19. Lyse the cells by sonication using a 5-second on, 5-second off cycle at 50% power for a cumulative time of 6 min.
SECTION: Clarification by Centrifugation
20. Centrifuge the lysate at 17000 rpm, 45 min to clarify.

SECTION: Protein Purification on Ni-NTA Column
21. Apply the supernatant to a Ni-NTA column (3x).
SECTION: Protein Purification on Ni-NTA Column
22. Wash the column until the wash buffer is free of protein, as measured by a Bradford assay.
SECTION: Protein Purification on Ni-NTA Column
23. Elute the protein using a wash buffer containing 300 millimolar (mM) imidazole.
SECTION: Size Exclusion Chromatography
24. Concentrate the eluted protein.
SECTION: Size Exclusion Chromatography
25. Apply the concentrated protein to an S75 10/300 column in buffer containing 25 millimolar (mM) HEPES pH 7.5, 150 millimolar (mM) NaCl, 1 millimolar (mM) MgCl2, and 1 millimolar (mM) TCEP.
SECTION: Final Protein Concentration
26. Concentrate the purified protein to a final concentration of 50 micromolar (µM).
SECTION: Final Protein Concentration
27. Divide the protein into 50 µL aliquots.
SECTION: Cryopreservation
28. Flash freeze the protein aliquots in liquid nitrogen.
SECTION: Cryopreservation
29. Store the frozen protein aliquots at -80 °C for future use.