a-Synuclein protein expression and purification

Thaw down an aliquot of plasmid construct (pRK172) encoding WT-human-a-synuclein 0.3mg/mL On ice

Thaw down On ice an aliquot of BL21 (DE3) RIL competent E Coli cells

Add 1µL of plasmid construct to the thawed competent cells and gently mix by flicking the bottom of the tube with a finger a few times

Incubate the reaction mix On ice

Perform heat-shock transformation 42°C in water bath incubator with manually shaking at100rpm

Equipment

Value	Label
Precision™ General Purpose Water Bath	NAME
Water Bath	TYPE
Thermo Scientific	BRAND
TSGP10	SKU
https://www.fishersci.com/us/en/home.html	LINK

Immediately transfer the tube on ice and incubate for 1 min.

Add 1000µL of SOC media to a chilled reaction

Incubate the bacteria 200rpm

Equipment

Value	Label
ThermoMixer® C	NAME
ThermoMixer	TYPE
Eppendorf	BRAND
5382000023	SKU

Prepare sterile 10cm LB agar plate containing 0.1mg/mL of ampicillin

Centrifuge at 500x g,10°C and discard the supernatant leaving 50µL of media

Spread 50 ul of cell suspension onto a selection plate and incubate overnight at 37°C in bacterial incubator

Equipment

Value	Label
Isotemp™ Microbiological Incubator, 178 L, Stainless Steel	NAME
Microbiological Incubator	TYPE
Fisherbrand	BRAND
15-103-0513	SKU
https://www.fishersci.com/us/en/home.html	LINK

Pick one colony and transfer into 10mL LB media with 0.1mg/mL of ampicillin

start in the morning (9:00 am)

Incubate the bacteria 250rpm until it reaches OD 0.2-0.3

Equipment

Value	Label
Natural convection incubator	NAME
Bacterial shaker	TYPE
Innova	BRAND
M1335-0000	SKU
https://www.eppendorf.com	LINK

Transfer a starter culture to 2X2L flasks filled with 0.5L LB media with 0.1mg/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)

Induce protein expression by adding 0.05millimolar (mM) IPTG, incubate at 18°C for 12h 0m 0sovernight

Collect the pellets by centrifugation (JA14 rotor) at 5000x g,0h 0m 0sx g at 4°C for 0h 10m 0s. Used 250 ml Beckman tubes

Usually get 10-12 g from 2L

Add to pellets 80 ml of lysis buffer (total): 10millimolar (mM) ThisHCl 7.6, 750millimolar (mM) NaCl, 1millimolar (mM) EDTA, 1millimolar (mM) PMSF (add just before using, have aliq frozen 0.1Molarity (M)), protease inhibitors (use MAXI version, need only one tablet);

Carefully resuspend the pellets to homogenize the solution

Heat up 1Lof 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 0h 1m 0s, 30%, 0h 0m 15s ON 0h 0m 30s OFF of amplitude then go to next falcon, had 3 falcons (repeat 3 times, avoid overheating)

After sonication samples need to get boiled thereby put the falcon tubes into glass beaker and boil for 0h 25m 0s. Use tweezers to pull out the tubes

Transfer boiled homogenates into new 50 mL falcon tubes; chill down suspensions at room temperature for 20 min

Prepare 4L of buffer 10millimolar (mM) TrisHCl 7.6, 50millimolar (mM) NaCl, 1millimolar (mM) EDTA, 1millimolar (mM) PMSF for dialysis

Centrifuge the homogenates at 20000x g,0h 0m 0sfor 1h 0m 0s at 4°C

Filter the supernatant using 0.45 um filter unit

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 into4Lplastic beaker filled with dialysis buffer, incubate overnight on magnetic plate on the slow mode (Chromatography fridge)

Equipment

Value	Label
ÄKTA pure 25 L1	NAME
ÄKTA pure chromatography system	TYPE
Cytiva	BRAND
29018225	SKU
https://www.cytivalifesciences.com/en/us	LINK

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);

Wash the column 2V of miliQ degassed water

Wash the column with 2V of STARTING BUFFER 10millimolar (mM) TrisHCl 7.6, 50millimolar (mM) NaCl

Activate with 1V of 10millimolar (mM) TrisHCl 7.6, 1Molarity (M) NaCl

Equilibrate with 3V of starting buffer

Load 80mL of suspension and then washed with 100 ml 50millimolar (mM)

NaCl 10millimolar (mM) TrisHCL, 300mLof 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 (10millimolar (mM) TrisHCl 7.6, 1Molarity (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 of each fraction with of 2X laemmli buffer and analyze fractions by SDS-PAGE with 4–20% gradient gels, followed by coomassie staining/destaining 10µL of each fraction with 10µL 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 4Lof 10millimolar (mM) TrisHCl 7.6, 50millimolar (mM) NaCl (follow instruction for dialysis)

Repeat section 'Protein purification (anion-exchange chromatography)' for the further fractionation of the purified preparation

Concentrate dialyzed protein sample to approximately 30mg/mL aliquot

Prepare the ultra-concentration system

Use 50 mL ultra centrifugation units with 3K cutoff

Wash off the unit with miliQ water through centrifugation at 5000x g,0h 0m 0s at 4°C for 0h 5m 0s , JA10 rotor

Load first 15mL of the sample into ultracentrifugation unit (max load of the unit is approx. 15mL)

Centrifuge at 5000x g,0h 0m 0s at 4°C for 0h 5m 0s , JA10 rotor

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

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

Follow instructions for with modifications

For a more successful endotoxin removal, add 1mL of

before the regeneration process

Collect the eluate into 5mL endotoxin-free tube and save 2 aliquots (10µLand 50µL) for protein concentration and endotoxin measurements

Follow instructions for Endotoxin detection kit LAL Genscript