Feb 16, 2024
Human and mouse alpha-synuclein protein expression and purification
Forked from a-Synuclein protein expression and purification
- 1Duke University
- West lab protocols
Protocol Citation: andrew.west west, Arpine Sokratian 2024. Human and mouse alpha-synuclein protein expression and purification. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm3rz9l3p/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 well
Created: February 16, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 95360
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020527
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Abstract
The protocol for is designed for high-yield purification of recombinant α-synuclein monomer. It is recommended to always store the protein on ice, and once the purification process has started, it should not be stopped.
Materials
BL21-Gold (DE3) Competent CellsAgilent TechnologiesCatalog #230132
Nalgene™ Rapid-Flow™ Sterile Single Use Vacuum Filter UnitsThermo Fisher ScientificCatalog #565-0010
SnakeSkin Dialysis Tubing 3.5K MWCO 35Thermo Fisher ScientificCatalog #88244
Endotoxin detection kit LAL GenscriptCatalog #95045-024
ToxinEraser™ Endotoxin Removal KitGenscriptCatalog #89233-330
ToxinEraser™ Endotoxin Removal ResinGenscriptCatalog #L00402
HiPrep Q HP anion exchange chromatography columnCytivaCatalog #29018182 MilliporeSigma™ Amicon™ Ultra-15 Centrifugal Filter UnitsCatalog #MilliporeSigma™ UFC901024
Protocol materials
Nalgene™ Rapid-Flow™ Sterile Single Use Vacuum Filter UnitsThermo Fisher ScientificCatalog #565-0010
SnakeSkin Dialysis Tubing 3.5K MWCO 35Thermo Fisher ScientificCatalog #88244
HiPrep Q HP anion exchange chromatography columnCytivaCatalog #29018182
MilliporeSigma™ Amicon™ Ultra-15 Centrifugal Filter UnitsCatalog #MilliporeSigma™ UFC901024
ToxinEraser™ Endotoxin Removal KitGenscriptCatalog #89233-330
ToxinEraser™ Endotoxin Removal ResinGenscriptCatalog #L00402
Endotoxin detection kit LAL GenscriptCatalog #95045-024
BL21-CodonPlus (DE3)-RILAgilent TechnologiesCatalog #230245-41
1.5mL Micro Centrifuge Tube; endotoxin-freeCELLTREATCatalog #50-202-024
ToxinEraser™ Endotoxin Removal KitGenscriptCatalog #89233-330
ToxinEraser™ Endotoxin Removal ResinGenscriptCatalog #L00402
HiPrep Q HP anion exchange chromatography columnCytivaCatalog #29018182
MilliporeSigma™ Amicon™ Ultra-15 Centrifugal Filter UnitsCatalog #MilliporeSigma™ UFC901024
BL21-Gold (DE3) Competent CellsAgilent TechnologiesCatalog #230132
Nalgene™ Rapid-Flow™ Sterile Single Use Vacuum Filter UnitsThermo Fisher ScientificCatalog #565-0010
SnakeSkin Dialysis Tubing 3.5K MWCO 35Thermo Fisher ScientificCatalog #88244
Endotoxin detection kit LAL GenscriptCatalog #95045-024
SOC MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #S1797-10X5ML
Ampicillin sodium saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0166
Agar powderGraingerCatalog #31FZ34
LB Broth, Miller (Granulated)Fisher ScientificCatalog #BP9723-500
IPTGResearch Products International Corp (RPI)Catalog #I56000-5.0
SODIUM CHLORIDEFisher ScientificCatalog #S2711
EDTA, disodium salt, dihydrateFisher ScientificCatalog #S312-500
cOmplete™, EDTA-free Protease Inhibitor Cocktail MIDI Merck MilliporeSigma (Sigma-Aldrich)Catalog #4693132001
Tris Base Research Products International Corp (RPI)Catalog #T60040-5000.0
Transformation
Transformation
1d
1d
Thaw down an aliquot of plasmid construct (pRK172) encoding WT-human-a-synuclein or mouse-a-synuclein 0.3 mg/mL On ice
15m
Thaw down On ice an aliquot of BL21 (DE3) RIL competent E Coli cells
BL21-CodonPlus (DE3)-RILAgilent TechnologiesCatalog #230245-41
15m
Add 1 µL of plasmid construct to the thawed competent cells and gently mix by flicking the bottom of the tube with a finger a few times
Safety information
do not resuspend
Incubate the reaction mix On ice
15m
Perform heat-shock transformation 42 °C in water bath incubator with manually shaking at100 rpm, 00:00:45
Equipment
Precision™ General Purpose Water Bath
NAME
Water Bath
TYPE
Thermo Scientific
BRAND
TSGP10
SKU
1m
Immediately transfer the tube on ice and incubate for 1 min.
1m
Add 1000 µL of SOC media to a chilled reaction
SOC MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #S1797-10X5ML
10s
Incubate the bacteria 200 rpm, 37°C, 00:30:00
Equipment
ThermoMixer® C
NAME
ThermoMixer
TYPE
Eppendorf
BRAND
5382000023
SKU
30m
Prepare sterile 10cm LB agar plate containing 0.1 mg/mL of ampicillinAmpicillin sodium saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0166
Agar powderGraingerCatalog #31FZ34
Collect 50 ul of cell suspension (Tube #1 5% of cells)
Centrifuge 950 cell suspension at 500 x g, 10°C, 00:03:00
Save the pellet with approximately 50 µL of media (Tube #2 95% of cells)
3m
Spread tubes #1 and #2 onto a selection plate and incubate overnight at 37 °C in bacterial incubator
Equipment
Isotemp™ Microbiological Incubator, 178 L, Stainless Steel
NAME
Microbiological Incubator
TYPE
Fisherbrand
BRAND
15-103-0513
SKU
10m
Protein expression
Protein expression
12h
12h
Pick one colony and transfer into 10 mL LB media with 0.1 mg/mL of ampicillin
start in the morning (9:00 am)
LB Broth, Miller (Granulated)Fisher ScientificCatalog #BP9723-500
Incubate the bacteria 250 rpm, 37°C, 05:00:00 until it reaches OD 0.2-0.3
Equipment
Natural convection incubator
NAME
Bacterial shaker
TYPE
Innova
BRAND
M1335-0000
SKU
5h
Transfer a starter culture to 2X2L flasks filled with 0.5L LB media with 0.1 mg/mL of ampicillin
5 mL to each flask
Equipment
New Brunswick™ Innova® 42
NAME
Incubated Benchtop Shakers
TYPE
Eppendorf
BRAND
M1335-0004
SKU
Incubate the culture at the same conditions until it reaches OD 0.8 (use nanodrop or cuvette) (reaches optimal density at 6-7 pm)
5h
Induce protein expression by adding 0.05 millimolar (mM) IPTG, incubate at 18 °C for 12:00:00 overnight
IPTGResearch Products International Corp (RPI)Catalog #I56000-5.0
Note
To cool down the grown culture, transfer the flasks into ice-bath and incubate until it reaches desired temperature
12h
Cell lysis
Cell lysis
11m 45s
11m 45s
Collect the pellets by centrifugation (JA14 rotor) at 5000 x g x g at 4 °C for 00:10:00 . Used 250 ml Beckman tubes
Usually get 10-12 g from 2L
Equipment
Avanti® J-E Centrifuge
NAME
Avanti Centrifuge
TYPE
Beckman
BRAND
369005
SKU
10m
Add to pellets 80 ml of lysis buffer (total): 10 millimolar (mM) ThisHCl 7.6 , 750 millimolar (mM) NaCl, 1 millimolar (mM) EDTA, 1 millimolar (mM) PMSF (add just before using, have aliq frozen 0.1 Molarity (M) ), protease inhibitors (use MAXI version, need only one tablet);
SODIUM CHLORIDEFisher ScientificCatalog #S2711 Tris Base Research Products International Corp (RPI)Catalog #T60040-5000.0
EDTA, disodium salt, dihydrateFisher ScientificCatalog #S312-500
cOmplete™, EDTA-free Protease Inhibitor Cocktail MIDI Merck MilliporeSigma (Sigma-Aldrich)Catalog #4693132001
Carefully resuspend the pellets to homogenize the solution
Heat up 1 L of water in a high temperature resistant glass beaker (turn heat to the max on the magnetic stirrer)
While waiting on water to get to the boiling point sonicate the lysates (use thick prob-tip) for 00:01:00 , 30%, 00:00:15 ON 00:00:30 OFF of amplitude then go to next falcon, had 3 falcons (repeat 3 times, avoid overheating)
10m
After sonication samples need to get boiled thereby put the falcon tubes into glass beaker and boil for 00:25:00 . Use tweezers to pull out the tubes
25m
Transfer boiled homogenates into new 50 mL falcon tubes; chill down suspensions at room temperature for 20 min
20m
Prepare 4 L of buffer 10 millimolar (mM) TrisHCl 7.6 , 50 millimolar (mM) NaCl, 1 millimolar (mM) EDTA, 1 millimolar (mM) PMSF for dialysis
Centrifuge the homogenates at 20000 x g for 01:00:00 at 4 °C
1h
Filter the supernatant using 0.45 um filter unit Nalgene™ Rapid-Flow™ Sterile Single Use Vacuum Filter UnitsVWR International (Avantor)Catalog #565-0010
Transfer filtered supernatant into dialysis bag which is: SnakeSkin Dialysis tubing, 3.5K MWCO, 35 mm dry I.D., 35 feet.
Measure the dialysis tube taking into consideration that 5 cm length of tube holds 48 mL of the sample (plus 2.5cm at each end for closure). Clip the tube using green clips, make sure it does not leak.
Place the dialysis bag into4 L plastic beaker filled with dialysis buffer, incubate overnight on magnetic plate on the slow mode (Chromatography fridge)
SnakeSkin Dialysis Tubing 3.5K MWCO 35VWR International (Avantor)Catalog #88244
Equipment
ÄKTA pure 25 L1
NAME
ÄKTA pure chromatography system
TYPE
Cytiva
BRAND
29018225
SKU
Protein purification (anion-exchange chromatography)
Protein purification (anion-exchange chromatography)
After a night of dialysis (4 °C slow mixing) collect the suspension into 100 mL glass bottle (filter the sample before running on the column, 0.22 um filter).
Column - HiPrep Q HP 16/10 column 1x20 ml (stored in 70% ethanol);HiPrep Q HP anion exchange chromatography columnVWR International (Avantor)Catalog #29018182
Wash the column 2V of miliQ degassed water
Wash the column with 2V of STARTING BUFFER 10 millimolar (mM) TrisHCl 7.6 , 50 millimolar (mM) NaCl
Activate with 1V of 10 millimolar (mM) TrisHCl 7.6 , 1 Molarity (M) NaCl
Equilibrate with 3V of starting buffer
Load 80 mL of suspension and then washed with 100 ml 50 millimolar (mM)
NaCl 10 millimolar (mM) TrisHCL, 300 mL of gradient elution (0-100%), 2 ml/min flow rate. Collected samples using fraction collector 2, every fraction 4 ml (use 10 ml glass tubes)
Place supernatant into channel A1 (was previously use for starting buffer, do not generate bubbles)
Place starting buffer in channel A2 (clean the tubing using the program mode)
Place elution buffer in channel B1 (10 millimolar (mM) TrisHCl 7.6 , 1 Molarity (M) NaCl)
Collected samples using fraction collector 2, every fraction 4 ml (use 10 ml glass tubes);
Analyze the fractions eluted at 250-350 mM salt (20 RFU conductivity) though SDS-PAGE (stain with Coomassie).
Combine 10 µL of each fraction with 10 µL of 2X laemmli buffer and analyze fractions by SDS-PAGE with 4–20% gradient gels, followed by coomassie staining/destaining
Measure A280/260 for the fractions containing single a-syn band, avoid collecting samples with A280/260 > 0.85
Combine the evaluated factions and measure total protein concentration using nanodrop.
Dialyze with 4 L of 10 millimolar (mM) TrisHCl 7.6 , 50 millimolar (mM) NaCl (follow instruction for dialysis)
Further purification
Further purification
Repeat section 'Protein purification (anion-exchange chromatography)' for the further fractionation of the purified preparation
Protein concentration
Protein concentration
10m
10m
Concentrate dialyzed protein sample to approximately 30 mg/mL aliquot
Add 3 µL of 10x diluted aliquot in PBS onto nanodrop pedestal;
Parameters:
- other proteins; coefficient extinction: 5.98; MW: 14.4 kDA (for wild-type human a-synuclein)
- other proteins; coefficient extinction: 7.45; MW: 14.4 kDA (for wild-type mouse a-synuclein)
Perform two measurements and confirm <10% standard error between two measurements
If necessary, prepare 20X and 30X dilutions to confirm findings.
Equipment
NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer
NAME
UV-Vis Spectrophotometer
TYPE
Thermo Scientific
BRAND
ND-ONE-W
SKU
Prepare the ultra-concentration system
Use 50 mL ultra centrifugation units with 3K cutoffMilliporeSigma™ Amicon™ Ultra-15 Centrifugal Filter UnitsVWR International (Avantor)Catalog #MilliporeSigma™ UFC901024
Wash off the unit with miliQ water through centrifugation at 5000 x g at 4 °C for 00:05:00 , JA10 rotor
5m
Load first 15 mL of the sample into ultracentrifugation unit (max load of the unit is approx. 15 mL )
Centrifuge at 5000 x g at 4 °C for 00:05:00 , JA10 rotor
5m
Resuspend concentrated sample, add more of protein sample and concentrate until the total volume is ~5 mL
Store at -80 °C . Yield should be approximately 80 mg per 2 L culture
Endotoxin removal
Endotoxin removal
Follow instructions for ToxinEraser™ Endotoxin Removal KitVWR International (Avantor)Catalog #89233-330 with modifications
For a more successful endotoxin removal, add 1 mL of
ToxinEraser™ Endotoxin Removal ResinVWR International (Avantor)Catalog #L00402 before the regeneration process
Collect the eluate into 5 mL endotoxin-free tube and save 2 aliquots (10 µL and 50 µL ) for protein concentration and endotoxin measurements
1.5mL Micro Centrifuge Tube; endotoxin-freeCELLTREATCatalog #50-202-024
Endotoxin quantification
Endotoxin quantification
Follow instructions for Endotoxin detection kit LAL GenscriptEndotoxin detection kit LAL VWR International (Avantor)Catalog #95045-024