Oct 01, 2023

Public workspaceProduction of GTPase Deficient RAB1A(Q70L) Protein

DOI
dx.doi.org/10.17504/protocols.io.5jyl8pe77g2w/v1
  • 1University of California, Berkeley;
  • 2ASAP CRN
Annan SI Cook
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DOI: dx.doi.org/10.17504/protocols.io.5jyl8pe77g2w/v1
Protocol Citation: Annan SI Cook 2023. Production of GTPase Deficient RAB1A(Q70L) Protein. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8pe77g2w/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: September 18, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 88628
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000350
Abstract
Bacterial expression of the GTPase deficient Q70L mutant of human RAB1A.
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Materials
Materials and Reagents

  • BL21 strain of E. coli
  • pET vector containing 6x-His-RAB1A(Q70L)
  • Ampicillin-resistant plates
  • LB broth
  • Isopropyl β-D-1-thiogalactopyranoside (IPTG)
  • Phosphate Buffered Saline (PBS)
  • Lysis buffer
  • EDTA-free protease inhibitor tablet (Thermo Fisher)
  • Phenylmethylsulfonyl fluoride (PMSF)
  • Ni-NTA column
  • Wash buffer (containing 300 mM imidazole)
  • S75 10/300 column
  • Liquid nitrogen

Lysis buffer
AB
HEPES pH 7.525 mM
NaCl300 mM
MgCl22 mM
TCEP1 mM
Transformation and Colony Selection
Transformation and Colony Selection
Transform E. coli BL21 cells with the pET vector containing 6x-His-RAB1A(Q70L) by heat shock.
Plate the transformed cells on ampicillin-resistant plates.
Incubate the plates DurationOvernight at Temperature37 °C .
Incubation
Overnight
Pre-culture and Expansion
Pre-culture and Expansion
Inoculate a single colony into Amount10 mL of LB broth.

Incubate the culture DurationOvernight at Temperature37 °C .

Incubation
Overnight
Main Culture Preparation
Main Culture Preparation
Transfer the entire pre-culture into a 1 L culture flask.
Grow the Amount1 L culture at Temperature37 °C with shaking at Shaker220 rpm until the optical density (OD) reaches approximately 0.6.

Cooling and Induction
Cooling and Induction
15m
15m
Place the flask in an ice bath for Duration00:15:00 to cool rapidly.

15m
Induce protein expression by adding Concentration0.1 millimolar (mM) IPTG.

Continue incubating the culture DurationOvernight (approximately 16 hours) at Temperature18 °C with shaking at Shaker180 rpm .
Incubation
Overnight
Cell Harvest and Pelleting
Cell Harvest and Pelleting
Harvest the cells by centrifugation at Centrifigation4000 rpm in a BECKMAN Coulter centrifuge.

Centrifigation
Cell Washing and Freezing
Cell Washing and Freezing
Wash the cell pellet once with PBS.
Wash
Pellet the cells again at Centrifigation4000 x g .

Centrifigation
Flash freeze the pellet in liquid nitrogen.
Store the frozen pellet at Temperature-80 °C until purification.

Temperature
Cell Lysis
Cell Lysis
6m
6m
Thaw the frozen cell pellet to TemperatureRoom temperature .

Resuspend the cells in lysis buffer.

Lysis buffer
AB
HEPES pH 7.525 mM
NaCl300 mM
MgCl22 mM
TCEP1 mM

Add an EDTA-free protease inhibitor tablet (Thermo Fisher) and PMSF to a concentration of Concentration1 millimolar (mM) .
Lyse the cells by sonication using a 5-second on, 5-second off cycle at 50% power for a cumulative time of Duration00:06:00 .
6m
Clarification by Centrifugation
Clarification by Centrifugation
45m
45m
Centrifuge the lysate at Centrifigation17000 rpm, 00:45:00 to clarify.

45m
Centrifigation
Protein Purification on Ni-NTA Column
Protein Purification on Ni-NTA Column
Apply the supernatant to a Ni-NTA column (3x).
Wash the column until the wash buffer is free of protein, as measured by a Bradford assay.
Wash
Elute the protein using a wash buffer containing Concentration300 millimolar (mM) imidazole.
Size Exclusion Chromatography
Size Exclusion Chromatography
Concentrate the eluted protein.
Apply the concentrated protein to an S75 10/300 column in buffer containing Concentration25 millimolar (mM) HEPES Ph7.5 , Concentration150 millimolar (mM) NaCl, Concentration1 millimolar (mM) MgCl2, and Concentration1 millimolar (mM) TCEP.
Final Protein Concentration
Final Protein Concentration
Concentrate the purified protein to a final concentration of Concentration50 micromolar (µM) .
Divide the protein into Amount50 µL aliquots.
Cryopreservation
Cryopreservation
Flash freeze the protein aliquots in liquid nitrogen.
Store the frozen protein aliquots at Temperature-80 °C for future use.

Temperature