Jan 15, 2024

Public workspacea-Synuclein protein expression and purification

  • 1Duke University
Open access
Protocol Citationandrew.west west, Arpine Sokratian 2024. a-Synuclein protein expression and purification. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4brwrvo5/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: May 21, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 62989
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020527
Abstract
Protocol for recombinant a-synuclein purification, useful as monomer template for seeded-amplification assays (like RT_QUIC or PMCA) . Recommendations are to store the protein always on ice while not running and do not stop purification when it is started.
Materials
1. BL21-CodonPlus (DE3)-RIL Chemical Competent Cells (Agilent #230245-41)
2. Thermo Scientific™ Low Protein Binding Collection Tubes (1.5 mL) PI90411

ReagentNalgene™ Rapid-Flow™ Sterile Single Use Vacuum Filter UnitsThermo Fisher ScientificCatalog #565-0010
ReagentSnakeSkin Dialysis Tubing 3.5K MWCO 35Thermo Fisher ScientificCatalog #88244
ReagentEndotoxin detection kit LAL GenscriptCatalog #95045-024

ReagentToxinEraser™ Endotoxin Removal KitGenscriptCatalog #89233-330
ReagentToxinEraser™ Endotoxin Removal ResinGenscriptCatalog #L00402
ReagentHiPrep Q HP anion exchange chromatography columnCytivaCatalog #29018182 ReagentMilliporeSigma™ Amicon™ Ultra-15 Centrifugal Filter UnitsCatalog #MilliporeSigma™ UFC901024

Protocol materials
ReagentToxinEraser™ Endotoxin Removal ResinGenscriptCatalog #L00402
Materials, Step 41
ReagentHiPrep Q HP anion exchange chromatography columnCytivaCatalog #29018182
Materials, Step 24
ReagentMilliporeSigma™ Amicon™ Ultra-15 Centrifugal Filter UnitsCatalog #MilliporeSigma™ UFC901024
Materials, Step 35
ReagentNalgene™ Rapid-Flow™ Sterile Single Use Vacuum Filter UnitsThermo Fisher ScientificCatalog #565-0010
Materials, Step 21
ReagentSnakeSkin Dialysis Tubing 3.5K MWCO 35Thermo Fisher ScientificCatalog #88244
Materials, Step 22
ReagentEndotoxin detection kit LAL GenscriptCatalog #95045-024
Materials, Step 43
ReagentToxinEraser™ Endotoxin Removal KitGenscriptCatalog #89233-330
Materials, Step 41
Transformation
Transformation
1d
Thaw down an aliquot of plasmid construct (pRK172) encoding WT-human-a-synuclein Concentration0.3 mg/mL TemperatureOn ice

15m
Thaw down TemperatureOn ice an aliquot of BL21 (DE3) RIL competent E Coli cells

15m
Add Amount1 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


Pipetting
Incubate the reaction mix TemperatureOn ice

15m
Incubation
Perform heat-shock transformation Temperature42 °C in water bath incubator with manually shaking atShaker100 rpm, 00:00:45
Equipment
Precision™ General Purpose Water Bath
NAME
Water Bath
TYPE
Thermo Scientific
BRAND
TSGP10
SKU



1m
Mix
Immediately transfer the tube on ice and incubate for 1 min.
1m
Add Amount1000 µL of SOC media to a chilled reaction

10s
Incubate the bacteria Shaker200 rpm, 37°C, 00:30:00
Equipment
ThermoMixer® C
NAME
ThermoMixer
TYPE
Eppendorf
BRAND
5382000023
SKU


30m
Prepare sterile 10cm LB agar plate containing Concentration0.1 mg/mL of ampicillin

Centrifuge at Centrifigation500 x g, 10°C, 00:03:00 and discard the supernatant leaving Amount50 µL of media

3m
Spread 50 ul of cell suspension onto a selection plate and incubate overnight at Temperature37 °C in bacterial incubator
Equipment
Isotemp™ Microbiological Incubator, 178 L, Stainless Steel
NAME
Microbiological Incubator
TYPE
Fisherbrand
BRAND
15-103-0513
SKU

10m
Overnight
Protein expression
Protein expression
12h
Pick one colony and transfer into Amount10 mL LB media with Concentration0.1 mg/mL of ampicillin
start in the morning (9:00 am)
Incubate the bacteria Shaker250 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 Concentration0.1 mg/mL of ampicillin
5 mL to each flask
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 Concentration0.05 millimolar (mM) IPTG, incubate at Temperature18 °C for Duration12:00:00 overnight

Note
To cool down the grown culture, transfer the flasks into ice-bath and incubate until it reaches desired temperature


12h
Pause
Overnight
Cell lysis
Cell lysis
11m 45s
Collect the pellets by centrifugation (JA14 rotor) at Centrifigation5000 x g x g at Temperature4 °C for Duration00:10:00 . Used 250 ml Beckman tubes
Usually get 10-12 g from 2L
10m
Add to pellets 80 ml of lysis buffer (total): Concentration10 millimolar (mM) ThisHCl Ph7.6 , Concentration750 millimolar (mM) NaCl, Concentration1 millimolar (mM) EDTA, Concentration1 millimolar (mM) PMSF (add just before using, have aliq frozen Concentration0.1 Molarity (M) ), protease inhibitors (use MAXI version, need only one tablet);

Carefully resuspend the pellets to homogenize the solution
Heat up Amount1 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 Duration00:01:00 , 30%, Duration00:00:15 ON Duration00:00:30 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 Duration00:25:00 . 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 Amount4 L of buffer Concentration10 millimolar (mM) TrisHCl Ph7.6 , Concentration50 millimolar (mM) NaCl, Concentration1 millimolar (mM) EDTA, Concentration1 millimolar (mM) PMSF for dialysis

Centrifuge the homogenates at Centrifigation20000 x g for Duration01:00:00 at Temperature4 °C

1h
Filter the supernatant using 0.45 um filter unit ReagentNalgene™ Rapid-Flow™ Sterile Single Use Vacuum Filter UnitsThermo Fisher ScientificCatalog #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 intoAmount4 L plastic beaker filled with dialysis buffer, incubate overnight on magnetic plate on the slow mode (Chromatography fridge)
ReagentSnakeSkin Dialysis Tubing 3.5K MWCO 35Thermo Fisher ScientificCatalog #88244

Equipment
ÄKTA pure 25 L1
NAME
ÄKTA pure chromatography system
TYPE
Cytiva
BRAND
29018225
SKU


Overnight
Protein purification (anion-exchange chromatography)
Protein purification (anion-exchange chromatography)
After a night of dialysis (Temperature4 °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);ReagentHiPrep Q HP anion exchange chromatography columnCytivaCatalog #29018182



Wash the column 2V of miliQ degassed water
Wash the column with 2V of STARTING BUFFER Concentration10 millimolar (mM) TrisHCl Ph7.6 , Concentration50 millimolar (mM) NaCl
Activate with 1V of Concentration10 millimolar (mM) TrisHCl Ph7.6 , Concentration1 Molarity (M) NaCl
Equilibrate with 3V of starting buffer
Load Amount80 mL of suspension and then washed with 100 ml Concentration50 millimolar (mM)
NaCl Concentration10 millimolar (mM) TrisHCL, Amount300 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 (Concentration10 millimolar (mM) TrisHCl Ph7.6 , Concentration1 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 Amount10 µL of each fraction with Amount10 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 Amount4 L of Concentration10 millimolar (mM) TrisHCl Ph7.6 , Concentration50 millimolar (mM) NaCl (follow instruction for dialysis)

Overnight
Further purification
Further purification
Repeat section 'Protein purification (anion-exchange chromatography)' for the further fractionation of the purified preparationGo togo to step #23

Overnight
Protein concentration
Protein concentration
10m
Concentrate dialyzed protein sample to approximately Concentration30 mg/mL aliquot
Prepare the ultra-concentration system
Use 50 mL ultra centrifugation units with 3K cutoffReagentMilliporeSigma™ Amicon™ Ultra-15 Centrifugal Filter UnitsCatalog #MilliporeSigma™ UFC901024

Wash off the unit with miliQ water through centrifugation at Centrifigation5000 x g at Temperature4 °C for Duration00:05:00 , JA10 rotor

5m
Load first Amount15 mL of the sample into ultracentrifugation unit (max load of the unit is approx. Amount15 mL )

Centrifuge at Centrifigation5000 x g at Temperature4 °C for Duration00:05:00 , JA10 rotor

5m
Resuspend concentrated sample, add more of protein sample and concentrate until the total volume is ~Amount5 mL

Store at Temperature-80 °C . Yield should be approximately Amount80 mg per 2 L culture

Pause
Endotoxin removal
Endotoxin removal
Follow instructions for ReagentToxinEraser™ Endotoxin Removal KitGenscriptCatalog #89233-330 with modifications
For a more successful endotoxin removal, add Amount1 mL of
ReagentToxinEraser™ Endotoxin Removal ResinGenscriptCatalog #L00402 before the regeneration process

Collect the eluate into Amount5 mL endotoxin-free tube and save 2 aliquots (Amount10 µL and Amount50 µL ) for protein concentration and endotoxin measurements

Endotoxin quantification
Endotoxin quantification
Follow instructions for Endotoxin detection kit LAL GenscriptReagentEndotoxin detection kit LAL GenscriptCatalog #95045-024