TITLE: Expression and purification of untagged asynuclein
AUTHORS: Alain Ndayisaba

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the generation of untagged a-synuclein.</div></div>

[STEPS]
?. [Expression]
Thaw - of BL21(DE3) competent E. coli  for ~ or until melted.
20 µl
50 µl
on ice
If more than   of cells are to be thawed, the remaining aliquot can be frozen using a 100% ethanol and dry ice bath.
?. [Expression]
Once the cells are completely thawed, add - of - of pET21a-alpha-synuclein and gently mix by inverting the tube.
1 µl
5 µl
10 pg
100 ng
Our lab has had consistent success using ~50-ng of pET21a-alpha-synuclein DNA for transformation.
?. [Expression]
Following the addition of the plasmid, incubate the cell and plasmid mixture  for about  .
on ice
This time can be reduced to a minimum of  , but the transformation efficiency significantly decreases with each halving of the time.
?. [Expression]
Then, heat-shock the cell and plasmid mixture for exactly  in a  water bath.
42 °C
?. [Expression]
Remove the mixture from the water bath and place it  for , then add - of SOC medium to the tube.
on ice
950 µl
980 µl
SOC medium is ideal, however LB medium can also be used but will result in a roughly two fold loss of transformation efficiency.
?. [Expression]
Incubate the bacteria at  for , while shaking at ~. After incubation, repeatedly invert the tube to mix the culture well and then plate  onto selection plates. Incubate the plate(s) upside-down overnight at  .
37 °C
Centrifuge: 250 33
100 µl
37 °C
Note: When first plating the transformants, is it recommended that different volumes of the culture (e.g.  ,  ,  ) are plated to ensure that at least 5-6 single colonies grow on each plate, but not so many that picking one becomes challenging.Note: If the plasmid contains resistance for ampicillin, the outgrowth step is not necessary[6]. However, if different resistance genes are present in the expression vector, it is necessary to perform this step to allow for cell recovery and expression of the antibiotic resistance gene(s).
?. [Expression]
On the following day, choose a single colony to inoculate in ~ of LB medium at  (pre-culture).
180 mL
37 °C
?. [Expression]
The next day, add a 1:20 dilution of the pre-culture to LB medium and grow at  until OD600 = 0.5-0.6. Once an OD600 = 0.5-0.6 is reached, induce the expression of αS with  IPTG and let the culture grow at  for ~.
37 °C
37 °C
The amount of IPTG to be used for the induction (typically  - ) depends on the cells, construct and culture conditions. It is recommended to perform a quick optimization, when setting up the expression protocol, in order to determine the optimal amount of IPTG. Also, it is not necessary to grow the cells at a lower temperature (e.g.  - ) because overexpressed αS does not tend to form inclusion bodies in E. coli due to its high solubility in the bacterial[17].
?. [Expression]
Then, spin the culture at  for  (). After the spin, remove the supernatant and freeze the pellet  at . Freezing the pellet in this fashion already lyses most of the bacteria.
Centrifuge: 9000 34
30 Room temperature
-20 °C
?. [Purification]
Cell Lysis:1. Pre-heat a hot plate and a  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 ( TRIS,  NaCl,  EDTA, ) and vortex until the pellet is completely resuspended, taking extra care not to leave any pellet in solution.
This protocol is written for an ÄKTAprime plus FPLC system equipped with a   UV and conductivity detectors. Although other FPLC systems will largely follow the same protocol, it is advised to always refer to the manufacturer’s specifications.
2 L
If cells are spun in a   centrifuge tube, approximately   of IEX buffer A should be enough to fully submerge the pellet.
?. [Purification]
2. Pour the resuspended cell suspension into the pre-heated  Erlenmeyer flask. Increase the hotplate temperature and let the mixture rise to an even boil.
2 L
?. [Purification]
3. After the first visible signs of boiling, allow the mixture to boil for an additional  to denature and precipitate proteases and other protein contaminants.
?. [Purification]
4. After boiling, let the lysate cool down to , transfer it to  centrifuge bottles and spin it at  for  (). Filtrate the supernatant through a  or  filter. Bring the supernatant up to approximately  with IEX buffer A.
4 °C
250 mL
Centrifuge: 20000 34
4 °C
0.22
0.45
300 mL
It is highly recommended to incubate the lysate mixture at   after cooling on the benchtop to avoid damaging centrifuge tubes upon high-speed centrifugation.
?. [Purification]
Ion Exchange Chromatography (IEX):1. Wash the FPLC system with IEX Buffer A at .
5
?. [Purification]
2. Connect the columns (2x  HiTrap Q HP columns) in the presence of a flow rate of  to ensure that no air enters the column. After the columns are attached, equilibrate them with IEX Buffer A at  for  or until the conductivity reaches a steady measurement for ~.
5 mL
0.3
1
If columns are stored in 20% ethanol it is recommended to wash the columns with MilliQ before equilibrating the column with buffer. This will ensure that salts will not precipitate in the column.
?. [Purification]
3. Following equilibration, load the cell lysate mixture onto the columns  at a max flow rate of .
1
?. [Purification]
4. Wash the column with IEX Buffer A until a steady (around ) plateau in the 280-nm absorbance is achieved, in order to remove any weakly bound contaminants.
40 mL
The wash step can also be performed using low percentages of IEX buffer B (e.g. 5-10%), in order to have more stringent wash conditions. However, this can also wash off small amounts of αS and should thus be tested by checking the waste of the washing step by Coomassie-stained SDS-PAGE or WB. In total, the volume of IEX Buffer A used during the wash step should be between  - .
?. [Purification]
5. After the UV absorbance plateaus, elute the protein with a gradient, from 0% to 100%, of IEX Buffer B ( TRIS,  NaCl,  EDTA, ) over  and collect fractions every .
75 mL
5 mL
?. [Purification]
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.
For a lab-scale expression (typically 1- or 2-L expression), 2x 5 mL HiTrap columns should be enough to allow for complete aS binding. If a larger expression is required, it is recommended to increase the number of HiTrap columns in the system or switch to a column(s) with a larger bed volume(s). Always analyze the flow-through fraction to ensure complete binding of aS to the columns. If aS is detected in the flow-through fraction, increase the number of columns in the system to increase the overall binding capacity.
?. [Purification]
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 , which will then be loaded on a size-exclusion column.
10 mL
?. [Purification]
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  of  ammonium acetate are run through the loop, switch the valve position back to “Load”.
25 mL
?. [Purification]
Size-Exclusion Chromatography (SEC):1. Wash the FPLC system with  ammonium acetate,  (.). Attach the SEC column (HiPrep 26/60 Sephacryl S-200 HR) in the presence of a buffer flow rate of ., to ensure that no air enters the column.
5
0.2
?. [Purification]
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.
Injecting small amounts of air into the columns is unavoidable (especially when working with a   sample loop). Air bubbles, when eluting, will give off characteristic spikes in the UV absorbance trace[20]. On the other hand, if large amounts of air are injected onto the column (i.e. resin discoloration from the presence of air is visible in the bed), generously flush the system and column with buffer. This applies to both IEX columns and SEC columns, but SEC columns are much  more sensitive to the presence of air in their resin because of their resin packing being critical for resolution (and cracks or lacunae can disrupt it completely and require column re-packing).
?. [Purification]
2. Attach a  loop to the six-way valve of the FPLC system and flush it with  of  ammonium acetate to remove any aggregated protein or built-up waste.
10 mL
20 mL
?. [Purification]
5. Flush a total of  of Ammonium Acetate buffer through the FPLC, collecting 14x  fractions between  and  corresponding to a molecular weight of 60 kDa.
350 mL
50 mL
110 mL
180 mL
?. [Purification]
8. After lyophilization, seal the tubes with Parafilm, to prevent moisture from entering, and store at  for short-term storage (1-2 weeks) or  for long-term storage.
4 °C
-20 °C
?. [Purification]
7. Aliquot the pooled fractions into  aliquots, then flash-freeze them using liquid nitrogen or a dry ice/ethanol bath and lyophilize (or store frozen at ) the samples.
1 mg
-80 °C
?. [Purification]
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.