TITLE: Purification of α-synuclein from E. coli
AUTHORS: Felix Kraus, vtrinkaus , Ruben Fernandez Busnadiego

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details methods for the purification of α-synuclein from E. coli – adapted from Volpicelly-Daley, Nat Protoc 2014.</div></div>

[STEPS]
?. [Purification of α-synuclein from E. coli]
Transform plasmid pT7-7 α-synuclein (α-syn, WT or A53T mutant) into BL21(DE3) cells (Agilent).
pT7-7 plasmids contain an Ampicillin (Amp) resistance. We thank Hilal Lashuel for depositing the plasmids on Addgene (Addgene, #105727 and #36046).
?. [Purification of α-synuclein from E. coli]
Grow an  culture (ONC) of transformed BL21(DE3) in lysogeny broth (LB) medium supplemented with  at .
[Ampicillin]
37 °C
?. [Purification of α-synuclein from E. coli]
Add  to , containing .
[overnight culture (ONC)]
[terrific broth (TB) medium]
[Ampicillin]
?. [Purification of α-synuclein from E. coli]
Incubate at  until an OD600 of 0.8 to 1.0 is reached.
37 °C
?. [Purification of α-synuclein from E. coli]
Induce protein expression with  for  at  (see Figure 1).
[IPTG]
37 °C
?. [Purification of α-synuclein from E. coli]
After 5 h of induction, spin the bacteria at  and freeze the resulting pellet at .
Centrifuge: 4000 34, 30 min
-20 °C
?. [Purification of α-synuclein from E. coli]
On the next day, thaw bacterial pellet .
on ice
?. [Purification of α-synuclein from E. coli]
Resuspend the bacterial pellet in high salt buffer containing , , , and protease inhibitor tablet (cOmplete, Roche - use 50 ml high salt buffer for 1 l of bacterial culture).
[NaCl]
[Tris, pH 7.6]
[EDTA]
?. [Purification of α-synuclein from E. coli]
Sonicate the bacterial suspension with a probe tip of at least 0.25 inch at 60 % power for  at .
[ (30 sec pulse on, 30 sec pulse off)]
4 °C
?. [Purification of α-synuclein from E. coli]
Boil the sonicated bacterial suspension for .
We use a glass beaker with boiling water. To avoid spilling of hot bacterial suspension, transfer suspension after sonication into 50 ml reaction tubes that contain holes in the lid, put those reaction tubes into the boiling water. Holes can be created with knifes or scissors.
?. [Purification of α-synuclein from E. coli]
After proteins precipitate during the boiling step (this takes roughly 15 to 20 min), cool the suspension .
on ice
?. [Purification of α-synuclein from E. coli]
As soon as bacterial suspension is cooled, spin it at  (make sure that all solid material is in the sediment and the supernatant is clear).
Centrifuge: 6000 34, 20 min
?. [Purification of α-synuclein from E. coli]
Take the supernatant and dialyze it  in , , .
[Tris, pH 7.6]
[NaCl]
[EDTA]
Make sure to use a sufficiently large volume for dialysis – for 200 ml protein solution, we use 5 l of dialysis buffer.
?. [Purification of α-synuclein from E. coli]
The next day, concentrate the dialyzed protein with a 3.5 kDa MWCO Amicon Filter device until the desired volume is reached (max. 5 ml for a 16/60 Superdex column, max. 12 ml for a 26/60 column).
?. [Purification of α-synuclein from E. coli]
Filter the protein solution through a syringe filter (0.22 µm). This avoids the loading of aggregated material onto the column. Load the filtered solution onto a Superdex 200 column (we recommend using a 26/60 column to allow the loading of larger volumes) and run it with the size exclusion buffer (, , ).
[Tris, pH 7.6]
[NaCl]
[EDTA]
?. [Purification of α-synuclein from E. coli]
Start collecting 1.5 ml fractions as soon as the void volume of the column flowed through. α-Syn typically behaves like a 50 kDa protein (collecting 96 fractions for a 26/60 column is usually sufficient).
?. [Purification of α-synuclein from E. coli]
Run a gel of every third fraction collected. Stain and destain, and identify fractions containing α-syn (we use precast gradient gels from Biorad, with a gradient ranging from 4 – 20 % to resolve small proteins) (Figure 2).
?. [Purification of α-synuclein from E. coli]
Pool α-syn containing fractions.
?. [Purification of α-synuclein from E. coli]
Dialyze pooled fraction  in , , .
[Tris, pH 7.6]
[NaCl]
[EDTA]
?. [Purification of α-synuclein from E. coli]
The next day, load protein solution onto a cation exchange column (e.g. a MonoQ, make sure that the NaCl concentration in the MonoQ washing buffer is low, so that α-syn can bind nicely to the column matrix).
?. [Purification of α-synuclein from E. coli]
Run a linear gradient ranging from 25 mM NaCl to 1 M NaCl (as described by VolpicellyDaley, Nat Protoc 2014.) on the MonoQ.
We found that one can omit the part of the gradient from 25 mM to 100 mM, and wash instead with 100 mM for one column volume. Additionally, the part of the gradient ranging from 600 mM to 1 M can be omitted as well. However, make sure to wash the column properly in the end to elute all proteins potentially bound to the column.
?. [Purification of α-synuclein from E. coli]
Collect  from 100 mM NaCl to 600 mM NaCl. α-Syn will elute at 300 mM NaCl (Figure 3).
[fractions]
?. [Purification of α-synuclein from E. coli]
Run a gel of all fractions, stain and destain, identify fractions containing α-syn.
?. [Purification of α-synuclein from E. coli]
Pool the fractions containing α-syn and dialyze  in , .
[Tris, pH 7.5]
[KCl]
?. [Purification of α-synuclein from E. coli]
On the next day, concentrate the protein solution to a final concentration of  (330 mM) and freeze it subsequently in batches of 1 ml at .
-80 °C
?. [Purification of α-synuclein from E. coli]
For conversion of α-syn into fibrils:
?. [Purification of α-synuclein from E. coli]
Thaw a tube with 1 ml α-syn monomer of a concentration of 5 mg/ml and centrifuge the solution at  (to pellet aggregates that might be present), take the supernatant and transfer it to a new 1 ml tube.
Centrifuge: 100000 34, 60 min
?. [Purification of α-synuclein from E. coli]
Shake at  in thermomixer.
37 33
?. [Purification of α-synuclein from E. coli]
Divide the fibrils into .
[aliquots]
We use PCR tubes; Fibrils can be stored at  .