Authors: Tim Bartels, Joanna Choi, Nora Kim & Dennis Selkoe
Pathogenic aggregation of α-Synuclein (αSyn) is implicated in familial and sporadic Parkinson disease (PD) and several other synucleinopathies. Choosing non-denaturing conditions, we developed a several purification schemes in order to obtain αSyn from human erythrocytes, enabling researchers to employ biophysical techniques in order to elucidate its native structure and properties.
Here we introduce a novel way of purifying human α-Synuclein from human erythrocytes, leaving the physiological conformation of the protein intact. This methology has been also successfully applied to human neuroblastoma cells stably overexpressing wt human α-Synuclein as well as to COS-7 cells transiently overexpressing wt and mutant forms of α-Synuclein. The overall purification is divided in three general steps: An ammonium sulfate preciptation giving a crude protein solution, a bulk purification which can be adjusted to the exact sample properties (volume, cell type, cross contamination) utilizing alternatively 3 different methods (AX, HIC, covalent chromatography), and a polishing step giving pure protein after size exclusion chromatography (Fig. 1).
Figure 1: Flow diagram of the purification scheme
Antibodies (usage according to manufacturers recommendations):
Ammonium sulfate precipitation:
Hydrophobic Interaction Chromatography (Äktapurifier and HiTrap Phenyl HP 5 ml)
Anion Exchange Chromatography (Äktapurifier and HitrapQ HP 5ml):
Covalent Chromatography (Äktapurifier and Thiopropyl Sepharose 6B packed in a 150 ml XK 16/100 column):
Wash column with 5 CV 20% EtOH ** Size Exclusion Chromatography (Superdex 200 10/300 GL)**:
Equilibrate column with 50 mM NH4Ac pH 8.5
Inject 250 ul of α-Synuclein positive concentrated fractions, collect fractions at flow rate 0.8 ml/min
Freeze/lyophilize peak fractions, take aliquot
A. Instead of freshly drawn blood, older samples can be employed. The ratio of α-Synuclein/Hemoglobin in those samples can be drastically reduced however, making the purification more challenging
A. It has to be made sure that the cell lysate does not heat up too much during sonication. Short sonication steps with incubation of the sample on ice in between are therefore recommended.
B. In cell culture cell lysates, a 25% (NH4)2SO4 pre-precipitation step can be beneficial to remove unwanted protein. If HIC is employed for the bulk purification, this is not necessary since the respective binding buffer already contains approx. 25% (NH4)2SO4 and thereby eliminates contamination before the column is loaded
Ammonium sulfate precipitation:
In erythrocyte lysate, washing the 50% (NH4)2SO4 pellet in 55% (NH4)2SO4 solution is highly recommended. Since Hemoglobin stays soluble until 60-65% (NH4)2SO4, the washing steps provide a cheap and easy method to eliminate large amounts of contaminating Hemoglobin.
The exact method of bulk purification has to be decided by the nature of the sample. In general, the HIC gave the highest purity but lowest yield. If high trough-put is needed, the Sepharose 6B method should be applied. If large amounts of Hemoglobin are present, AX is recommended. If a particular sample has low purity with one of the chosen methods, an additional step utilizing one of the other methods can be employed.
A: HIC advantages: Fast, cheap, best purity, (NH4)2SO4 in binding/elution buffer stabilizes folding. Disadvantages: Low binding capacity, struggles with high amounts of contaminating Hemoglobin. Results can be inconsistent depending on the exact sample. Has sometimes to be applied twice to effectively purifiy all α-Synuclein out of solution.
B: AX advantages: Fast, cheap, good binding affinity, Hemoglobin does not bind (making it very effective in separating α-Synuclein and Hemoglobin). Disadvantages: High amounts of NaCl in elution buffer can destabilize α-Synuclein, causing it to precipitate. Transthyretin and α-Synuclein elute very close to each other making thorough removal of blood plasma prior to erythrocyte lysing necessary. Has sometimes to be applied twice to effectively purifiy all α-Synuclein out of solution.
C: Instead of Sepharose 6B gel medium, Sepharose 4B can be used. Sepharose 6B has shown to have a better binding capacity, while Sepharose 4B packed columns can be easier reactivated. Sepharose 6B advantages: Simple and consistent, good for large sample volumes, high yield, removes both Transthyretin and Hemoglobin from erythrocyte lysate samples very effectively. Since no high pressures/high flow rates, no buffer gradient and no monitoring of UV-absorption or conductivity are needed, it can be done with a simple peristaltic pump. Disadvantages: Expensive gel media, no pre-packed columns commercially available, regeneration of column time consuming.
Size Exclusion Chromatography (Superdex 200):
Instead of a Superdex 200 column, Superdex 75, Superose 12 and Sephacryl 200-HR were also successfully employed. The advantage of the Superdex 200 gel medium is the ability to separate Hemoglobin from α-Synuclein in contrast to the above mentioned media.
From 40 ml of freshly drawn packed red blood cells, up to 0.5 mg α-Synuclein can be recovered.
As seen in Fig. 2, α-Synuclein usually precipitates between 30 and 45% (NH4)2SO4. Human Hemoglobin should stay soluble until 60%.
Figure 2: Ammonium sulfate precipitation of α-Synuclein from cell lysate
SDS-PAGE Western blot (C20) of α-Synuclein. Cell lysate (erythrocyte) was treated step wise with increasing (NH4)2SO4 concentrations, the resulting pellet was taken up in PBS and analyzed.
HIC (HiTrap Phenyl HP): α-Synuclein should elute between a conductivity of 135-110 mS/cm (~ approx. 0.7 M (NH4)2SO4) (Fig. 3A).
AX (HiTrap Q HP): α-Synuclein should elute between a conductivity of 30-35 mS/cm (~ approx. 300 mM NaCl) (Fig. 3B).
SEC (Superdex 200 10/300 GL): α-Synuclein should elute around 13.5 ml (Fig. 3C).
Figure 3: Example FPLC chromatograms of α-Synuclein purifications and WB analysis of fractions
Covalent chromatography (Sepharose 6B, XK 16/100): α-Synuclein should be completely found in the clear flow-trough. Hemoglobin should be completely bound to the column and then be eluted with DTT (Fig. 4).
Figure 4: Separation of Hemoglobin and α-Synuclein via covalent chromatography
Example Coomassie stains of the different purification steps for human erythrocytes are shown in Fig. 5. After applying the procedure the purity of the sample should be >95% (measured by densitometry of Coomassie stain).
Figure 5: Coomassie stain of different purification steps
SDS-PAGE Coomassie stain analysis of aliquots taken from different stages of purification.
α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation. Tim Bartels, Joanna G. Choi, and Dennis J. Selkoe. Nature doi:10.1038/nature10324
Tim Bartels, Joanna Choi, Nora Kim & Dennis Selkoe, Dennis Selkoe Lab
Source: Protocol Exchange (2011) doi:10.1038/protex.2011.254. Originally published online 15 August 2011.