Expression and purification of untagged asynuclein

Thaw 20µL-50µL of BL21(DE3) competent E. coli On ice for ~0h 10m 0s or until melted.

Once the cells are completely thawed, add 1µL-5µL of 10pg-100ng of pET21a-alpha-synuclein and gently mix by inverting the tube.

Following the addition of the plasmid, incubate the cell and plasmid mixture 37On ice for about 0h 30m 0s .

Then, heat-shock the cell and plasmid mixture for exactly 0h 0m 10s in a 42°C water bath.

Remove the mixture from the water bath and place it On ice for 0h 5m 0s, then add 950µL-980µL of SOC medium to the tube.

Incubate the bacteria at 37°C for 1h 0m 0s, while shaking at ~250rpm,0h 0m 0s. After incubation, repeatedly invert the tube to mix the culture well and then plate 100µL onto selection plates. Incubate the plate(s) upside-down overnight at 37°C .

On the following day, choose a single colony to inoculate in ~180mL of LB medium at 37°C 1h 0m 0s (pre-culture).

The next day, add a 1:20 dilution of the pre-culture to LB medium and grow at 37°C until OD₆₀₀ = 0.5-0.6. Once an OD₆₀₀ = 0.5-0.6 is reached, induce the expression of αS with 1millimolar (mM) IPTG and let the culture grow at 37°C for ~4h 0m 0s.

Then, spin the culture at 9000x g,0h 0m 0s for 0h 20m 0s (30Room temperature). After the spin, remove the supernatant and freeze the pellet 0h 20m 0s at -20°C. Freezing the pellet in this fashion already lyses most of the bacteria.

1. Pre-heat a hot plate and a 2L Erlenmeyer Flask to the minimum temperature to initiate boiling. While the hotplate and flask are reaching the right temperature (enough to bring the culture to a boil but not too high that it will cause charring), submerge the frozen pellet in IEX Buffer A (20millimolar (mM) TRIS, 25millimolar (mM) NaCl, 1millimolar (mM) EDTA, 8) and vortex until the pellet is completely resuspended, taking extra care not to leave any pellet in solution.

2. Pour the resuspended cell suspension into the pre-heated 2L Erlenmeyer flask. Increase the hotplate temperature and let the mixture rise to an even boil.

3. After the first visible signs of boiling, allow the mixture to boil for an additional 0h 15m 0s to denature and precipitate proteases and other protein contaminants.

4. After boiling, let the lysate cool down to 4°C, transfer it to 250mL centrifuge bottles and spin it at 20000x g,0h 0m 0s for 0h 45m 0s (4°C). Filtrate the supernatant through a 0.22μm or 0.45μm filter. Bring the supernatant up to approximately 300mL with IEX buffer A.

Ion Exchange Chromatography (IEX):

1. Wash the FPLC system with IEX Buffer A at 5ml/min.

2. Connect the columns (2x 5mL HiTrap Q HP columns) in the presence of a flow rate of 0.3ml/min to ensure that no air enters the column. After the columns are attached, equilibrate them with IEX Buffer A at 1ml/min for 0h 40m 0s or until the conductivity reaches a steady measurement for ~0h 20m 0s.

3. Following equilibration, load the cell lysate mixture onto the columns 0h 20m 0s at a max flow rate of 1ml/min.

4. Wash the column with IEX Buffer A until a steady (around 40mL) plateau in the 280-nm absorbance is achieved, in order to remove any weakly bound contaminants.

5. After the UV absorbance plateaus, elute the protein with a gradient, from 0% to 100%, of IEX Buffer B (20millimolar (mM) TRIS, 1Molarity (M) NaCl, 1millimolar (mM) EDTA, 8) over 75mL and collect fractions every 5mL.

6. Analyze all the fractions and the flow-through (the unbound lysate fraction which passes through the column and is collected in the FPLC waste during the lysate loading step) via Coomassie-stained SDS-PAGE or Western Blot (αS usually elutes between 25 and 35 mS) to determine the fractions with the highest concentration of αS.

7. Pool the fractions (usually around a conductivity of ~25 mSi when adopting this protocol and purification system) containing the greatest amount of αS and (eventually) concentrate down to 10mL, which will then be loaded on a size-exclusion column.

Size-Exclusion Chromatography (SEC):

1. Wash the FPLC system with 50millimolar (mM) ammonium acetate, 7.40 (5ml/min.). Attach the SEC column (HiPrep 26/60 Sephacryl S-200 HR) in the presence of a buffer flow rate of 0.2ml/min., to ensure that no air enters the column.

2. Attach a 10mL loop to the six-way valve of the FPLC system and flush it with 20mL of 50millimolar (mM) ammonium acetate to remove any aggregated protein or built-up waste.

3. Inject the pooled IEX fractions in the loop, making sure not to inject air bubbles into the system or column. The valve position must be set to “Load” when injecting the sample onto the loop.

4. At the beginning of the run, switch the valve position to “Inject”. At this point, the pooled IEX fractions will be injected onto the SEC column. After 25mL of 50millimolar (mM) ammonium acetate are run through the loop, switch the valve position back to “Load”.

5. Flush a total of 350mL of Ammonium Acetate buffer through the FPLC, collecting 14x 50mL fractions between 110mL and 180mL corresponding to a molecular weight of 60 kDa.

6. Analyze the fraction purity via Coomassie-stained SDS-PAGE and pool 5-6 of the purest fractions. Measure the absorbance of these fractions at 280 nm (ε(aS)=0.412 mL·mg-1·cm-1). Usually purity of >95% can be obtained.

7. Aliquot the pooled fractions into 1mg aliquots, then flash-freeze them using liquid nitrogen or a dry ice/ethanol bath and lyophilize (or store frozen at -80°C) the samples.

8. After lyophilization, seal the tubes with Parafilm, to prevent moisture from entering, and store at 4°C for short-term storage (1-2 weeks) or -20°C for long-term storage.